EA045580B1 - SYSTEMS AND METHODS FOR COORDINATING CELL PRODUCTION FOR INDIVIDUAL IMMUNOTHERAPY - Google Patents

Description

State of the art

Treatment of bulky, refractory cancers through adoptive transfer of tumor-infiltrating lymphocytes (TILs) represents a powerful therapeutic approach for patients with poor prognosis. Gattinoni, et al., Nat. Rev. Immunol. 2006, 6, 383-393. Successful immunotherapy requires large quantities of TILs, and commercialization requires an efficient and reliable process. This presented a challenge due to the technical, logistical and regulatory challenges associated with cell propagation. IL-2-based TIL propagation followed by the rapid expansion process (REP) has become the preferred method for TIL propagation due to its speed and efficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al., J. Immunother. 2003, 26, 332-42. REP can result in a 1000-fold expansion of TILs over a 14-day period, although this requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), often from several donors as feeder cells, as well as antibodies to CD3 (OCT3) and high doses of IL-2. Dudley, et al., J. Immunother. 2003, 26, 332-42. TILs subjected to the REP procedure provided successful adoptive cell therapy after host immunosuppression in melanoma patients.

Brief description of graphic materials

In fig. 1 schematically illustrates the various steps involved in treating a patient through adoptive cell therapy using TILs, including the various steps in the production of allogeneic TILs.

In fig. 2A shows the GEN 3 process timeline for TIL production.

In fig. 2B shows a comparison of Process 2A and the GEN 3 Process embodiment for TIL production.

In fig. 2C shows a comparison of an embodiment of the GEN 3 process, an embodiment of the GEN 3.1 process, and an alternative embodiment of the GEN 3.1 process for producing TIL.

In fig. 3A shows a block diagram of a TIL production coordination system for a patient.

In fig. 3B shows an object diagram for components of system 300 that are suitably modified or built upon commercially available software platforms in addition to standard components for those platforms, in accordance with some embodiments.

In fig. 3C-3E schematically illustrate tracking of biological material during production in a manufacturing facility in accordance with some embodiments.

In fig. 3F schematically illustrates the process of maintaining SOC and COI throughout the journey of a cell therapy product from solid tumor production through the manufacturing process to infusion into a patient in accordance with certain embodiments of the manufacturing process (eg, the GEN 3 process).

In fig. 3G is a representative image of a label for a cell therapy product in accordance with some embodiments.

In fig. 3H is a table showing various types of labels produced during the manufacturing process of a cell therapy product in accordance with some embodiments.

In fig. 3I and 3J are representative images of a finished product label in accordance with some embodiments.

In fig. ZK-ZR presents representative screenshots of tumor acquisition forms in accordance with some embodiments.

In fig. 4A and 4B show a flow chart for determining a patient's treatment event schedule based on the success of the TIL production process.

In fig. 4C shows a flow diagram for an alternative embodiment for determining a patient's treatment event schedule based on the success of the TIL production process.

In fig. 5 is a flowchart of Process Embodiment 2A, a 22-day TIL production process.

In fig. 6 shows a comparison of Process 1C and Process 2A embodiment for TIL production.

In fig. 7 shows the chronology of the 1C process.

In fig. 8 is a detailed diagram of process embodiment 2A.

In fig. 9 is an illustrative flow diagram of process 2A giving an overview of steps A through F.

In fig. 10 is a comparison table of steps A through F of exemplary embodiments of process 1C and process 2A.

In fig. 11 shows a detailed comparison between Process Embodiment 1C and Process Embodiment 2A.

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Brief description of the sequence listing

SEQ ID NO: 1 is the amino acid sequence of muromonab heavy chain.

SEQ ID NO: 2 is the amino acid sequence of muromonab light chain.

SEQ ID NO: 3 is the amino acid sequence of recombinant human IL2 protein.

SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

SEQ ID NO: 5 is a form of IL-2.

SEQ ID NO: 6 is the amino acid sequence of nemvaleukin alpha.

SEQ ID NO: 7 is a form of IL-2.

SEQ ID NO: 8 is a mucin domain polypeptide.

SEQ ID NO: 9 is the amino acid sequence of recombinant human IL4 protein.

SEQ ID NO: 10 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO: 11 is the amino acid sequence of recombinant human IL-15 protein.

SEQ ID NO: 12 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO: 13 is the sequence of IL-2.

SEQ ID NO: 14 is the sequence of IL-2 mutein.

SEQ ID NO: 15 is the sequence of IL-2 mutein.

SEQ ID NO: 16 is HCDR1IL-2 for IgG.IL2R67A.H1.

SEQ ID NO: 17 is HCDR2 for IgG.IL2R67A.H1.

SEQ ID NO: 18 is HCDR3 for IgG.IL2R67A.H1.

SEQ ID NO: 19 is Kabat's HCDR1_IL-2 for IgG.IL2R67A.H1.

SEQ ID NO: 20 is Kabat HCDR2 for IgG.IL2R67A.H1.

SEQ ID NO: 21 is Kabat HCDR3 for IgG.IL2R67A.H1.

SEQ ID NO: 22 is Chothia HCDR1_IL-2 for IgG.IL2R67A.H1.

SEQ ID NO: 23 is Chothia HCDR2 for IgG.IL2R67A.H1.

SEQ ID NO: 24 is Chothia HCDR3 for IgG.IL2R67A.H1.

SEQ ID NO: 25 is IMGT HCDR1_IL-2 for IgG.IL2R67A.H1.

SEQ ID NO: 26 is the HCDR2 IMGT for IgG.IL2R67A.H1.

SEQ ID NO: 27 is the HCDR3 IMGT for IgG.IL2R67A.H1.

SEQ ID NO: 28 is the V _H chain for IgG.IL2R67A.H1.

SEQ ID NO: 29 is the heavy chain for IgG.IL2R67A.H1.

SEQ ID NO: 30 is Kabat LCDR1 for IgG.IL2R67A.H1.

SEQ ID NO: 31 is Kabat LCDR2 for IgG.IL2R67A.H1.

SEQ ID NO: 32 is Kabat LCDR3 for IgG.IL2R67A.H1.

SEQ ID NO: 33 is Chothia LCDR1 for IgG.IL2R67A.H1.

SEQ ID NO: 34 is Chothia LCDR2 for IgG.IL2R67A.H1.

SEQ ID NO: 35 is Chothia LCDR3 for IgG.IL2R67A.H1.

SEQ ID NO: 36 is a V _L chain.

SEQ ID NO: 37 is a light chain.

SEQ ID NO: 38 is a light chain.

SEQ ID NO: 39 is a light chain.

SEQ ID NO: 40 is the amino acid sequence of human 4-1BB.

SEQ ID NO: 41 is the amino acid sequence of mouse 4-1BB.

SEQ ID NO: 42 is the heavy chain of the anti-41BB agonist monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 43 is the light chain of the anti-41BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 44 is the heavy chain variable region ( _VH ) of the anti-4-1BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 45 is the light chain variable region (V _L ) of the anti-4-1BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 46 is the heavy chain CDR1 of the anti-4-1BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 47 is the heavy chain CDR2 of the anti-4-1BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 48 is the heavy chain CDR3 of the anti-4-1BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 49 is CDR1 of the light chain of an agonistic monoclonal antibody

- 2 045580 to 4-1BB utomilumab (PF-05082566).

SEQ ID NO: 50 is the light chain CDR2 of the anti-4-1BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 51 is the light chain CDR3 of the anti-4-1BB agonistic monoclonal antibody utomilumab (PF-05082566).

SEQ ID NO: 52 is the heavy chain of the anti-41BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 53 is the light chain of the anti-41BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 54 is the heavy chain variable region (VH) of the 4-1BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 55 is the light chain variable region ( _VL ) of the anti-4-1BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 56 is the heavy chain CDR1 of the anti-4-1BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 57 is the heavy chain CDR2 of the anti-4-1BB agonistic monoclonal antibody utomilumab (BMS-663513).

SEQ ID NO: 58 is the heavy chain CDR3 of the anti-4-1BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 59 is the light chain CDR1 of the 4-1BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 60 is the light chain CDR2 of the anti-4-1BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 61 is the light chain CDR3 of the 4-1BB agonistic monoclonal antibody urelumab (BMS-663513).

SEQ ID NO: 62 is the Fc domain of a TNFRSF agonist fusion protein.

SEQ ID NO: 63 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 64 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 65 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 66 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 67 is a linker for a TNFRSF agonist fusion protein.

SEQ ID NO: 68 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 69 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 70 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 71 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 72 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 73 is the Fc domain of a TNFRSF agonist fusion protein.

SEQ ID NO: 74 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 75 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 76 is a TNFRSF agonist fusion protein linker.

SEQ ID NO: 77 is the amino acid sequence of ligand 4-1BB (4-1BBL).

SEQ ID NO: 78 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO: 79 is the heavy chain variable region (VH) of the anti-4-1BB agonist antibody 4B4-1-1 version 1.

SEQ ID NO: 80 is the light chain variable region ( _VL ) of the anti-4-1BB agonist antibody 4B4-1-1 version 1.

SEQ ID NO: 81 is the heavy chain variable region (VH) of the anti-4-1BB agonist antibody 4B4-1-1 version 2.

SEQ ID NO: 82 is the light chain variable region ( _VL ) of the anti-4-1BB agonist antibody 4B4-1-1 version 2.

SEQ ID NO: 83 is the heavy chain variable region ( _VH ) of the anti-4-1BB agonist antibody H39E3-2.

SEQ ID NO: 84 is the light chain variable region (V _L ) of the anti-4-1BB agonist antibody H39E3-2.

SEQ ID NO: 85 is the amino acid sequence of human OX40.

SEQ ID NO: 86 is the amino acid sequence of mouse OX40.

SEQ ID NO: 87 is the heavy chain of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 88 is the light chain of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 89 is the heavy chain variable region (VH) of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

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SEQ ID NO: 90 is the light chain variable region ( _VL ) of the OX40 agonistic monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 91 is the heavy chain CDR1 of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 92 is the heavy chain CDR2 of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 93 is the heavy chain CDR3 of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 94 is the light chain CDR1 of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 95 is the light chain CDR2 of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 96 is the light chain CDR3 of the agonistic anti-OX40 monoclonal antibody tavolixizumab (MEDI-0562).

SEQ ID NO: 97 is the heavy chain of the agonistic anti-OX40 monoclonal antibody 11D4.

SEQ ID NO: 98 is the light chain of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 99 is the heavy chain variable region (VH) of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 100 is the light chain variable region (VL) of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 101 is the heavy chain CDR1 of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 102 is the heavy chain CDR2 of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 103 is the heavy chain CDR3 of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 104 is the light chain CDR1 of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 105 is the light chain CDR2 of the agonistic anti-OX40 monoclonal antibody 11D4.

SEQ ID NO: 106 is the light chain CDR3 of the OX40 agonistic monoclonal antibody 11D4.

SEQ ID NO: 107 is the heavy chain of the agonistic anti-OX40 monoclonal antibody 18D8.

SEQ ID NO: 108 is the light chain of the OX40 agonistic monoclonal antibody 18D8.

SEQ ID NO: 109 is the heavy chain variable region ( _VH ) of the OX40 agonistic monoclonal antibody 18D8.

SEQ ID NO: 110 is the light chain variable region (V _L ) of the OX40 agonistic monoclonal antibody 18D8.

SEQ ID NO: 111 is the heavy chain CDR1 of the agonistic anti-OX40 monoclonal antibody 18D8.

SEQ ID NO: 112 is the heavy chain CDR2 of the agonistic anti-OX40 monoclonal antibody 18D8.

SEQ ID NO: 113 is the heavy chain CDR3 of the OX40 agonistic monoclonal antibody 18D8.

SEQ ID NO: 114 is the light chain CDR1 of the agonistic anti-OX40 monoclonal antibody 18D8.

SEQ ID NO: 115 is the light chain CDR2 of the OX40 agonistic monoclonal antibody 18D8.

SEQ ID NO: 116 is the light chain CDR3 of the OX40 agonistic monoclonal antibody 18D8.

SEQ ID NO: 117 is the heavy chain variable region (VH) of the agonistic anti-OX40 monoclonal antibody Hu119-122.

SEQ ID NO: 118 is the light chain variable region (VL) of the agonistic anti-OX40 monoclonal antibody Hu119-122.

SEQ ID NO: 119 is the heavy chain CDR1 of the agonistic anti-OX40 monoclonal antibody Hu119-122.

SEQ ID NO: 120 is the heavy chain CDR2 of the OX40 agonistic monoclonal antibody Hu119-122.

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SEQ ID NO: 121 is the CDR3 of the heavy chain of the OX40 agonistic monoclonal antibody Hu119-122.

SEQ ID NO: 122 is the light chain CDR1 of the OX40 agonistic monoclonal antibody Hu119-122.

SEQ ID NO: 123 is the light chain CDR2 of the agonistic anti-OX40 monoclonal antibody Hu119-122.

SEQ ID NO: 124 is the light chain CDR3 of the OX40 agonistic monoclonal antibody Hu119-122.

SEQ ID NO: 125 is the heavy chain variable region (VH) of the agonistic anti-OX40 monoclonal antibody Hu106-222.

SEQ ID NO: 126 is the light chain variable region (V _L ) of the agonistic anti-OX40 monoclonal antibody Hu106-222.

SEQ ID NO: 127 is the heavy chain CDR1 of the OX40 agonistic monoclonal antibody Hu106-222.

SEQ ID NO: 128 is the heavy chain CDR2 of the OX40 agonistic monoclonal antibody Hu106-222.

SEQ ID NO: 129 is the heavy chain CDR3 of the agonistic anti-OX40 monoclonal antibody Hu106-222.

SEQ ID NO: 130 is the light chain CDR1 of the agonistic anti-OX40 monoclonal antibody Hu106-222.

SEQ ID NO: 131 is the light chain CDR2 of the OX40 agonistic monoclonal antibody Hu106-222.

SEQ ID NO: 132 is the light chain CDR3 of the OX40 agonistic monoclonal antibody Hu106-222.

SEQ ID NO: 133 is the amino acid sequence of OX40 ligand (OX40L).

SEQ ID NO: 134 is the soluble portion of the OX40L polypeptide.

SEQ ID NO: 135 is an alternative soluble portion of the OX40L polypeptide.

SEQ ID NO: 136 is the heavy chain variable region (VH) of the agonistic monoclonal antibody to OX40008.

SEQ ID NO: 137 is the light chain variable region ( _VL ) of an agonistic monoclonal antibody to OX40008.

SEQ ID NO: 138 is the heavy chain variable region (VH) of the agonistic monoclonal antibody to OX40011.

SEQ ID NO: 139 is the light chain variable region ( _VL ) of an agonistic monoclonal antibody to OX40011.

SEQ ID NO: 140 is the heavy chain variable region (VH) of the agonistic monoclonal antibody to OX40021.

SEQ ID NO: 141 is the light chain variable region ( _VL ) of the agonistic monoclonal antibody to OX40021.

SEQ ID NO: 142 is the heavy chain variable region (VH) of the agonistic monoclonal antibody to OX40023.

SEQ ID NO: 143 is the light chain variable region ( _VL ) of the agonistic monoclonal antibody to OX40023.

SEQ ID NO: 144 is the heavy chain variable region (VH) of an agonistic monoclonal antibody to OX40.

SEQ ID NO: 145 is the light chain variable region (VL) of an OX40 agonistic monoclonal antibody.

SEQ ID NO: 146 is the heavy chain variable region (VH) of an agonistic monoclonal antibody to OX40.

SEQ ID NO: 147 is the light chain variable region (V _L ) of an OX40 agonistic monoclonal antibody.

SEQ ID NO: 148 is the heavy chain variable region (VH) of a humanized agonistic anti-OX40 monoclonal antibody.

SEQ ID NO: 149 is the heavy chain variable region (VH) of a humanized agonistic anti-OX40 monoclonal antibody.

SEQ ID NO: 150 is the light chain variable region (VL) of a humanized OX40 agonistic monoclonal antibody.

SEQ ID NO: 151 is the light chain variable region (VL) of a humanized OX40 agonistic monoclonal antibody.

SEQ ID NO: 152 is the heavy chain variable region (VH) of a humanized agonistic anti-OX40 monoclonal antibody.

SEQ ID NO: 153 is the heavy chain variable region (VH) of a humanized

- 5 045580 agonistic monoclonal antibody to OX40.

SEQ ID NO: 154 is the light chain variable region (V _L ) of a humanized agonistic monoclonal antibody to OX40.

SEQ ID NO: 155 is the light chain variable region (V _L ) of a humanized OX40 agonistic monoclonal antibody.

SEQ ID NO: 156 is the heavy chain variable region (VH) of an agonistic monoclonal antibody to OX40.

SEQ ID NO: 157 is the light chain variable region (V _L ) of an OX40 agonistic monoclonal antibody.

SEQ ID NO: 158 is the amino acid sequence of the heavy chain of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 159 is the amino acid sequence of the light chain of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 160 is the amino acid sequence of the heavy chain variable region ( _VH ) of the PD-1 inhibitor nivolumab.

SEQ ID NO: 161 is the amino acid sequence of the light chain variable region (V _L ) of the PD-1 inhibitor nivolumab.

SEQ ID NO: 162 is the amino acid sequence of the heavy chain CDR1 of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 163 is the CDR2 amino acid sequence of the heavy chain of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 164 is the CDR3 amino acid sequence of the heavy chain of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 165 is the amino acid sequence of the light chain CDR1 of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 166 is the amino acid sequence CDR2 of the light chain of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 167 is the amino acid sequence CDR3 of the light chain of nivolumab, a PD-1 inhibitor.

SEQ ID NO: 168 is the amino acid sequence of the heavy chain of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 169 is the amino acid sequence of the light chain of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 170 is the amino acid sequence of the heavy chain variable region (VH) of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 171 is the amino acid sequence of the variable light chain (VL) region of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 172 is the amino acid sequence of the heavy chain CDR1 of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 173 is the amino acid sequence of the heavy chain CDR2 of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 174 is the amino acid sequence of the heavy chain CDR3 of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 175 is the amino acid sequence of the light chain CDR1 of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 176 is the amino acid sequence CDR2 of the light chain of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 177 is the amino acid sequence CDR3 of the light chain of pembrolizumab, a PD-1 inhibitor.

SEQ ID NO: 178 is the heavy chain amino acid sequence of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 179 is the amino acid sequence of the light chain of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 180 is the amino acid sequence of the heavy chain variable region ( _VH ) of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 181 is the amino acid sequence of the light chain variable region (V _L ) of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 182 is the amino acid sequence of the heavy chain CDR1 of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 183 is the CDR2 amino acid sequence of the heavy chain of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 184 is the amino acid sequence CDR3 of the heavy chain of dur-6045580 valumab, a PD-L1 inhibitor.

SEQ ID NO: 185 is the amino acid sequence of the light chain CDR1 of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 186 is the amino acid sequence CDR2 of the light chain of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 187 is the amino acid sequence CDR3 of the light chain of durvalumab, a PD-L1 inhibitor.

SEQ ID NO: 188 is the amino acid sequence of the heavy chain of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 189 is the amino acid sequence of the light chain of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 190 is the amino acid sequence of the heavy chain variable region (VH) of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 191 is the amino acid sequence of the variable light chain (VL) region of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 192 is the amino acid sequence of the heavy chain CDR1 of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 193 is the amino acid sequence CDR2 of the heavy chain of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 194 is the amino acid sequence CDR3 of the heavy chain of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 195 is the amino acid sequence of the light chain CDR1 of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 196 is the amino acid sequence CDR2 of the light chain of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 197 is the amino acid sequence CDR3 of the light chain of avelumab, a PD-L1 inhibitor.

SEQ ID NO: 198 is the heavy chain amino acid sequence of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 199 is the amino acid sequence of the light chain of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 200 is the amino acid sequence of the heavy chain variable region (VH) of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 201 is the amino acid sequence of the light chain variable region (VL) of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 202 is the amino acid sequence of the heavy chain CDR1 of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 203 is the amino acid sequence of the heavy chain CDR2 of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 204 is the CDR3 amino acid sequence of the heavy chain of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 205 is the amino acid sequence of the light chain CDR1 of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 206 is the amino acid sequence of the light chain CDR2 of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 207 is the amino acid sequence CDR3 of the light chain of atezolizumab, a PD-L1 inhibitor.

SEQ ID NO: 208 is the amino acid sequence of the heavy chain of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 209 is the amino acid sequence of the light chain of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 210 is the amino acid sequence of the heavy chain variable region ( _VH ) of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 211 is the amino acid sequence of the variable light chain (VL) region of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 212 is the amino acid sequence of the heavy chain CDR1 of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 213 is the CDR2 amino acid sequence of the heavy chain of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 214 is the CDR3 amino acid sequence of the heavy chain of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 215 is the amino acid sequence of the light chain CDR1 of ipi-limumab, a CTLA-4 inhibitor.

SEQ ID NO: 216 is the amino acid sequence CDR2 of the light chain of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 217 is the amino acid sequence CDR3 of the light chain of ipilimumab, a CTLA-4 inhibitor.

SEQ ID NO: 218 is the amino acid sequence of the heavy chain of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 219 is the amino acid sequence of the light chain of tremelimu mab, a CTLA-4 inhibitor.

SEQ ID NO: 220 is the amino acid sequence of the heavy chain variable region (VH) of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 221 is the amino acid sequence of the variable light chain (VL) region of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 222 is the amino acid sequence of the heavy chain CDR1 of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 223 is the CDR2 amino acid sequence of the heavy chain of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 224 is the CDR3 amino acid sequence of the heavy chain of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 225 is the amino acid sequence of the light chain CDR1 of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 226 is the amino acid sequence CDR2 of the light chain of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 227 is the amino acid sequence CDR3 of the light chain of tremelimumab, a CTLA-4 inhibitor.

SEQ ID NO: 228 is the amino acid sequence of the heavy chain of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 229 is the amino acid sequence of the light chain of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 230 is the amino acid sequence of the heavy chain variable region ( _VH ) of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 231 is the amino acid sequence of the light chain variable region (VL) of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 232 is the amino acid sequence of the heavy chain CDR1 of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 233 is the heavy chain CDR2 amino acid sequence of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 234 is the heavy chain CDR3 amino acid sequence of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 235 is the amino acid sequence of the light chain CDR1 of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 236 is the light chain CDR2 amino acid sequence of zalifrelimab, a CTLA-4 inhibitor.

SEQ ID NO: 237 is the light chain CDR3 amino acid sequence of zalifrelimab, a CTLA-4 inhibitor.

Detailed description of the invention

I. Introduction.

Adoptive cell therapy using TILs cultured using the rapid expansion protocol (REP) has resulted in successful adoptive cell therapy following host immunosuppression in melanoma patients. For example, in some cases, lymphodepletion prior to adoptive transfer of tumor-specific T lymphocytes has been found to play a key role in increasing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (cytokine residues). In some cases, the effectiveness of ACT may be increased by pretreating the patient receiving ACT with nonmyeloablative chemotherapy prior to the TIL infusion, for example, 28 to 25 days before the TIL infusion. It has also been found that an IL-2 treatment regimen following TIL infusion may improve the chances of therapy success. The timing and duration of these procedures before and after the infusion determine the final effectiveness of the entire treatment regimen.

Thus, planning of different treatment regimens depends on the timing and duration of the TIL manufacturing process, which itself depends on the acceptability parameters of the final product. Current infusion acceptance parameters depend on the TIL formulation indication (eg, CD28, CD8, or CD4 positivity), as well as the expansion factor and viability of the expanded TIL product (also referred to herein as the REP product). In turn, the multiplicity

- 8 045580 propagation and viability of the REP product depend on various parameters measured during the propagation process. Such differences in yield and timing of obtaining an acceptable REP product for infusion pose logistical challenges in transporting the tumor to the manufacturing facility, transferring the REP product, and scheduling various patient treatment events during the manufacturing process.

TIL.

In addition, various parameters such as cell viability and cell number at different stages of the TIL production process determine the duration of subsequent steps so that at the end of the TIL production process, acceptable viability, numerical fold and final cell number are obtained.

Additionally, it is important to track biological material from the time it is removed from the patient to the time it is infused into the patient, including throughout the TIL manufacturing process, to avoid production delays, material mislabeling, and patient misidentification, thereby increasing patient safety.

The present invention provides a framework for coordinating the manufacturing process of an expanded cell therapy product for a patient and various patient treatment events by dynamically scheduling various patient treatment events based on various measured parameters at different stages of the manufacturing process.

For example, in one embodiment, a method of coordinating the production of expanded T cells for a patient may include obtaining, by computation, a request to order T cell expansion cells for the patient; generating, by the computing device, a patient-specific identifier or cell order identifier associated with the cell order request; and initiation of the T cell proliferation process. The T cell expansion process may involve performing a procedure on a patient in a healthcare facility to obtain T cells from the patient and transferring the resulting T cells to a manufacturing facility. Once the resulting T cells are received at the manufacturing facility, the patient's treatment events are dynamically scheduled by the computing device. Dynamic scheduling depends on the acceptance parameters of subsequently obtained T cell expansion. The expansion of T cells from at least some of the obtained T cells is initiated using a cell expansion technique and the parameters of acceptability of the expansion T cells are determined.

In another embodiment, a method of coordinating the production of an expanded cell therapy product for a patient may include receiving, through a computing device, a request to order cells for expanding the cell therapy product for the patient; generating, by the computing device, a patient-specific identifier including a cell order identifier associated with the cell order request; and initiating the propagation process of the cell therapy product. The process of propagating a cell therapy product may involve performing a procedure on a patient at a healthcare facility to obtain a solid tumor from the patient and transferring the resulting solid tumor to a manufacturing facility. Once a solid tumor is received in the manufacturing plant, the patient's treatment events are dynamically planned by the computing device. Dynamic planning depends on the acceptability parameters of the subsequently expanded cell therapy product. The propagation of the cell therapy product from at least a portion of the obtained solid tumor is initiated using a cell propagation technique, and the acceptability parameters of the propagated cell therapy product are determined at the first time point and at the second time point following the first time point. It is determined whether the acceptance parameters of the expanded cell therapy product meet certain acceptance criteria at the first time point and at the second time point. If the acceptance parameters at the first time point meet the acceptance criteria, expansion of the cell therapy product is continued until the second time point. If the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at the second time point, it is determined whether the cell therapy product can be expanded again using the cell expansion technique from the first time point based on the acceptance parameters at the second time point. If it is determined that reexpansion is possible, the cell therapy product is expanded from at least some of the cell therapy product obtained at the second time point using the cell expansion technique from the first time point. The time of completion of propagation of the cell therapy product is estimated after repeated propagation of the cell therapy product from the first time point. The patient's treatment event schedule is modified based on the estimated time of completion of expansion of the cell therapy product and the timing of treatment events before or after infusion of the expanded cell therapy product into the patient's body. Subsequent propagation of the cell therapy product is completed from the first time point.

The present invention further provides methods and systems for accurately tracking biological material from the time it is removed from a patient until it is reinfused into the patient. In particular, the methods and systems described herein facilitate the maintenance of a chain of identification and a chain of responsibility for biological material.

For example, in one embodiment, a method for tracking biological material from a patient may include receiving a request to order cells to produce biological material for the patient. Upon receiving a cell order request, the computing device generates a patient-specific identifier associated with the cell order request. The patient-specific identifier may include one or more of a patient identifier, a cell order request identifier, an order code, and a cell order lot number. The patient's biological material is then tracked during delivery of the biological material from the clinical site where the biological material is removed from the patient to the manufacturing facility, at the manufacturing facility during the manufacturing process, and during delivery of the manufactured biological material from the manufacturing facility to the clinical facility where the manufactured material is located. biological material, and back to the clinical facility, where the patient is infused with the manufactured biological material using a patient-specific identifier. Tracking may include recording, by a computing device, a tracking event at each stage of the delivery and production processes. The tracking event record includes the chain of custody of the patient's biological material.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention belongs. All patents and publications referenced herein are incorporated herein by reference in their entirety.

The term in vivo refers to an event occurring in the body of a subject.

The term in vitro refers to an event occurring outside the subject's body. In vitro assays include cell-based assays that use live or dead cells, and may also include cell-free assays that do not use intact cells.

The term ex vivo refers to an event that involves treating or performing a procedure on a cell, tissue and/or organ that has been removed from the body of the subject. Accordingly, the cell, tissue and/or organ may be returned to the subject by surgery or treatment.

The term rapid expansion means an increase in the number of antigen-specific TILs by at least about 3-fold (or 4, 5, 6, 7, 8, or 9-fold) within a week, more preferably at least about 10-fold (or 20, 30). , 40, 50, 60, 70, 80 or 90 times) over the course of a week, or most preferably at least about 100 times over the course of a week. Below are a number of rapid propagation protocols.

The term tumor-infiltrating lymphocytes or TILs as used herein refers to a population of cells originally derived as white blood cells that have left the subject's bloodstream and migrated into the tumor. TILs include, but are not limited to, cytotoxic CD8+ T cells (lymphocytes), Th1 and Th7 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. Primary TILs are those TILs that are derived from patient tissue samples as described herein (sometimes referred to as freshly obtained or freshly isolated), and secondary TILs are any populations of TIL cells that have been expanded or proliferated as described herein, including , among other things, total TILs and propagated TILs (REP TIL or post-REP TIL). TIL cell populations may include genetically modified TILs.

A population of cells (including TILs) as used herein refers to a number of cells that share common characteristics. Overall populations typically range from 1 x 10 ⁶ to 1 x 10 ¹⁰ , with different TIL populations comprising different amounts. For example, the initial growth of primary TILs in the presence of IL-2 results in a population of total TILs of approximately 1x10 ⁸ cells. REP propagation is typically carried out to provide populations of 1.5 x 10 ⁹ to 1.5 x 10 ¹⁰ cells for infusion. In some embodiments, REP propagation is performed to provide populations of 2.3 x 10 ¹⁰ to 13.7 x 10 ¹⁰ .

By cryopreserved TIL as used herein, it is meant that the TIL, either primary, core, or expanded (REP TIL), is processed and stored in the range of about -150°C to -60°C. General cryopreservation methods are also described elsewhere herein, including in the examples. For clarity, cryopreserved TILs are different from frozen tissue samples, which can be used as a source of primary TILs.

By thawed cryopreserved TILs, as used herein, we mean a population of TILs that have previously been cryopreserved and then processed to return to room temperature or above, including, but not limited to, cell culture temperatures or temperatures at which the TILs can be administered to a patient.

TILs can usually be defined either biochemically, using cell surface markers, or functionally, by their ability to invade tumors and influence treatment. TIL is usually possible

- 10 045580 classify by the expression of one or more of the following biomarkers: CD4, CD8, TCR ap,

CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1 and CD25. Additionally and alternatively, TILs can be functionally determined to infiltrate solid tumors when reintroduced to a patient.

The term cryopreservation media or cryopreservation medium refers to any medium that can be used for cryopreservation of cells. Such media may include media containing 7% to 10% DMSO. Exemplary environments include CryoStor CS10, Hyperthermasol, and combinations thereof. The term CS10 refers to cryopreservation media obtained from Stemcell Technologies or Biolife Solutions.

The CS10 media may be referred to under the trade name CryoStor® CS10. CS10 medium is a serum-free, animal-free medium containing DMSO.

The term central memory T cell refers to a subset of T cells that in humans are CD45R0+ and constitutively express CCR7 (CCR7 ^hl ) and CD62L (CD62 ^hl ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Central memory T cells primarily secrete IL-2 and CD40L as effector molecules upon TCR triggering. Central memory T cells predominate in the CD4 compartment in the blood, and in humans they are proportionately more abundant in the lymph nodes and tonsils.

The term effector memory T cells refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+ but have lost constitutive expression of CCR7 (CCR7 ^|0 ) and are heterogeneous or low in expression of CD62L (CD62L ^{| 0} ). The surface phenotype of central memory T cells also includes TCR, CD3, CD127 (IL-7R) and IL-15R. Transcription factors for central memory T cells include BLIMP1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines after antigenic stimulation, including interferon-γ, IL-4, and IL-5. Effector memory T cells predominate in the CD8 compartment in the blood, and in humans they are proportionately more abundant in the lungs, liver, and intestines. Effector memory CD8+ T cells carry large amounts of perforin.

The term closed system refers to a system that is closed to the external environment. Any closed system suitable for cell culture methods can be used with the methods of the present invention. Closed systems include, but are not limited to, for example, closed G containers. Once a tumor segment is added to a closed system, the system is not opened to the outside environment until the TILs are ready for administration to the patient.

The terms fragmentation, fragmentation, and fragmentation as used herein to describe tumor destruction processes include mechanical fragmentation methods such as crushing, cutting, fission, and morcellation of tumor tissue, as well as any other method of disrupting the physical structure of tumor tissue.

The term fine needle aspiration or FNA refers to a type of biopsy procedure that can be used for sampling or diagnostic procedures, including tumor sampling, in which a sample is taken but the tumor is not removed or resected. In fine needle aspiration, a hollow needle, such as a 25-18 gauge, is inserted into the tumor or area containing the tumor and fluid and cells (including tissue) are obtained for further analysis or expansion as described herein. In FNA, cells are removed without preserving the histological architecture of tissue cells.

FNA may contain TILs. In some cases, fine-needle aspiration biopsy is performed using an ultrasound-guided fine-needle aspiration biopsy needle. FNA needles are commercially available from Becton Dickinson, Covidien, etc.

The term core needle biopsy or core biopsy refers to a type of biopsy procedure that can be used for sampling or diagnostic procedures, including tumor sampling, in which a sample is taken but the tumor is not removed or resected. In a core needle biopsy, a hollow needle, such as a 16-11 gauge, is inserted into the tumor or area containing the tumor and fluid and cells (including tissue) are obtained for further analysis or expansion as described herein. With core needle biopsy, cells can be removed with some preservation of the histological architecture of tissue cells, given the larger needle size compared to FNA. The core biopsy needle is usually sized to preserve at least some of the histologic architecture of the tumor. Core needle biopsies may contain TILs. In some cases, core biopsy is performed using a core biopsy instrument, vacuum core biopsy instrument, stereotactic core biopsy instrument, ultrasound-guided core biopsy instrument, MRI-guided core biopsy instrument, commercially available from Bard Medical, Becton Dickinson and etc.

The terms peripheral blood mononuclear cells and PBMC refer to peripheral blood cells that have a round nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. When used as antigen presenting cells (PBMCs are a type

- 11 045580 antigen presenting cells) peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells.

The terms peripheral blood lymphocytes and PBL refer to T cells that proliferate from the peripheral blood. In some embodiments, PBL is isolated from whole blood or apheresis product of a donor. In some embodiments, PBL is isolated from whole blood or apheresis product of a donor by positive or negative selection for a T cell phenotype, such as a CD3 ⁺ CD45+ T cell phenotype.

The term anti-CD3 antibody refers to an antibody or variant thereof, for example, a monoclonal antibody, including human, humanized, chimeric or murine antibodies, that is directed against the CD3 receptor on the T-cell antigen receptor of mature T cells. Antibodies to CD3 include OKT-3, also known as muromonab. Antibodies to CD3 also include the UHCT1 clone, also known as T3 and CD3ε. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab and visilizumab.

The term OKT-3 (also referred to herein as OKT3) refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric or murine antibodies directed against the CD3 receptor in the mature T cell antigen receptor and includes commercially available forms , such as OKT-3 (30 ng/ml, pure MACS GMP CD3, Miltenyi Biotech, Inc., San Diego, CA, USA) and muromonab or its variants, conservative amino acid substitutions, glycoforms or biosimilars. The amino acid sequences of the heavy and light chains of muromonab are given in table. 1 (SEQ ID NO: 1 and SEQ ID NO: 2). The hybridoma capable of producing OKT-3 is deposited in the American Type Culture Collection and is assigned the public access number ATCC CRL 8001. The hybridoma capable of producing OKT-3 is also deposited in the European Collection of Authenticated Cell Cultures (ECACC) and is assigned the catalog number 86022706.

- 12 045580

Table 1

Amino acid sequences of muromonab

Identifier R Sequence (single letter amino acid symbols) SEQIDNO:1 Muromonab heavy chain QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60 NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120 KTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180 YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ GO NO:2 Muromonab light chain QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH 60 FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120 SEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180 TKDEYERHNS YTCEATHKTS TSPIVKSFNRNEC 213

The term IL-2 (also referred to herein as IL2) refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-2 is described, for example, in Nelson, J. Immunol. 2004, 172, 3983-88 and publications Malek, Annu. Rev. Immunol. 2008, 26, 453-79, the description of which is incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is presented in table. 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2 such as aldesleukin (PROLEUKIN, commercially available from several suppliers in 22 million IU dosages in single-dose vials), as well as the commercially available form of recombinant IL-2 from CellGenix, Inc. ., Portsmouth, NH, USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-209-b) and other commercial equivalents from other suppliers. Aldesleukin (des-alanyl-1, human IL-2 series-125) is a non-glycosylated recombinant form of human IL-2 with a molecular weight of approximately 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is presented in table. 2 (SEQ ID NO: 4). The term IL-2 also covers pegylated forms of IL-2 as described herein, including the pegylated IL2 prodrug bempegaldesleukin (NKTR-214, pegylated human recombinant IL-2 as in SEQ ID NO: 4, which has an average of 6 lysine residues is N ⁶ substituted with [(2,7-bis-{[methylpoly(oxyethylene)]carbamoyl}-9H-fluoren-9-yl)methoxy]carbonyl), which is available from Nektar Therapeutics, South San Francisco, NY California, USA, or which can be prepared by methods known in the art, such as the methods described in Example 19 of International Patent Application Publication No. WO 2018/132496 A1, or the method described in Example 1 of US Patent Application Publication No. US 2019/0275133 A1, description of

- 13 045580 which are incorporated herein by reference. Bempegaldesleukin (NKTR-214) and other pegylated IL-2 molecules suitable for use in the present invention are described in US Patent Application Publication No. US 2014/0328791 A1 and International Patent Application Publication No. WO 2012/065086 A1, the description of which is included incorporated into this document by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in US Patent Nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosure of which is incorporated herein by reference. IL-2 compositions suitable for use in the present invention are described in US Pat. No. 6,706,289, the disclosure of which is incorporated herein by reference.

In some embodiments, a form of IL-2 suitable for use in the present invention is THOR-707, available from Synthorx, Inc. The preparation and properties of THOR-707 and additional alternative forms of IL-2 suitable for use in the present invention are described in US Patent Application Publications Nos. US 2020/0181220 A1 and US 2020/0330601 A1, the disclosure of which is incorporated herein by reference. . In some embodiments, a form of IL-2 suitable for use in the present invention is an interleukin 2 (IL-2) conjugate comprising: an isolated and purified IL2 polypeptide; and a conjugating moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107, wherein the amino acid residue numbering corresponds to SEQ ID NO: 5. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some embodiments, the amino acid position is selected from R38 and K64. In some embodiments, the amino acid position is selected from E61, E62, and E68. In some embodiments, the amino acid position is at position E62. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to lysine, cysteine, or histidine. In some embodiments, the amino acid residue is mutated to cysteine. In some embodiments, the amino acid residue is mutated to lysine. In some embodiments, an amino acid residue selected from K35, T37, R38, T41, F42, K43, f44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107 is further mutated to an unnatural amino acid. In some embodiments, the unnatural amino acid comprises N6-azidoethoxy-L-lysine (AzK), N6-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornenelysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl-lysine, 2-amino-8- oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, pazidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8oxononanoic acid, p- propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-Dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-Lphenylalanine, p-benzoylA-phenylalanine, p-bromophenylalanine, p-aminoA -phenylalanine, isopropyl-Lphenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allylA-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine , phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-( phenylselanyl)propanoic or selenocysteine. In some embodiments, the IL-2 conjugate has reduced affinity for the IL-2 receptor subunit (IL-2Ra) compared to wild-type IL-2 polypeptide. In some embodiments, the reduction in affinity is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% reduction in binding affinity for IL- 2Ra versus wild-type IL-2 polypeptide. In some embodiments, the affinity reduction is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 500-fold, 1000-fold or more compared to wild-type IL-2 polypeptide. In some embodiments, the conjugating moiety disrupts or blocks the binding of IL-2 to IL-2Ra. In some embodiments, the conjugating moiety comprises a water-soluble polymer. In some embodiments, the additional conjugating moiety comprises a water-soluble polymer. In some embodiments, each of the water-soluble polymers independently comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), ethylene glycol-propylene glycol copolymers, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(ahydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(Nacryloylmorpholine) or combinations thereof. In some embodiments, each of the water-soluble polymers independently contains PEG. In some embodiments, the PEG is a linear PEG or a branched PEG. In some embodiments, each of the water

- 14 045580 soluble polymers independently contain a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, each of the water-soluble polymers independently contains a glycan. In some embodiments, each of the water-soluble polymers independently contains a polyamide. In some embodiments, the conjugating moiety comprises a protein. In some embodiments, the additional conjugating moiety comprises a protein. In some embodiments, each of the proteins independently comprises albumin, transferrin, or transthyretin. In some embodiments, each of the proteins independently contains an Fc portion. In some embodiments, each of the proteins independently contains the Fc portion of an IgG. In some embodiments, the conjugating moiety comprises a polypeptide. In some embodiments, the additional conjugating moiety comprises a polypeptide. In some embodiments, each of the polypeptides independently comprises an XTEN peptide, a glycine-rich homoamino acid polymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein (GLK) polymer. In some embodiments, the isolated and purified IL-2 polypeptide is modified by glutamylation. In some embodiments, the conjugating moiety is directly linked to the isolated and purified IL-2 polypeptide. In some embodiments, the conjugating moiety is indirectly linked to the isolated and purified IL-2 polypeptide through a linker. In some embodiments, the linker comprises a homobifunctional linker. In some embodiments, the homobifunctional linker comprises Lohmann's reagent dithiobis(succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo -DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobispropionimidate (DTBP), 1,4-di(3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide compound (DFDNB), e.g. 1,5-difluoro-2,4-dinitrobenzene or dinitrobenzene,

4,4'-difluoro-3,3'-dinitrophenylsulfone

1,3-difluoro-4,6bis-[b-(4(DFDNPS), azidosalicylamido)ethyl] disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine , 3,3'dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylenebis(iodoacetamide) or N,N'-hexamethylenebis(iodoacetamide). In some embodiments, the linker comprises a heterobifunctional linker. In some embodiments, the heterobifunctional linker comprises N-succinimidyl-3-(2-pyridyldithio)propionate (sPDP), a long-chain N-succinimidyl-3-(2-pyridyldithio)propionate (LC-sPDP), a water-soluble long-chain

N-succinimidyl-3-(2-pyridyldithio)propionate methyl-a-(2-pyridyldithio)toluene (sMPT), thio)toluamido]hexanoate (sulfo-LC-sMPT), (sulfo-LC-sPDP), succinimidyloxycarbonyl-αsulfosuccinimidyl -6-[a-methyl-a-(2-pyridyldisuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1carboxylate (sMCC), sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfosMCC), ester m -maleimidobenzoyl-N-hydroxysuccinimide (MB), mmaleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MB), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl -4-(pmaleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(γ-maleimidobutyryloxy)succinimide ester (GMB), N-(γ-maleimidobutyryloxy)sulfosuccinium deester ( sulfo-GMB), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6(((iodoacetyl)amino)hexanoyl)amino]hexanoate (slAXX), succinimidyl 4-(((iodoacetyl)amino) methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl reactive and sulfhydryl reactive crosslinking agents such as 4-(4-Nmaleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyhydrazide-8 (M2C2H),

3-(2-pyridyldithio)propionylhydrazide (PDPH),

N-hydroxysuccinimidyl-4azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido) hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2- (p-azidosalicylamido)ethyl 1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), Nsuccinimidyl-6-(4'-azido -2'-nitrophenylamino) hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANBNOs), sulfosuccinimidyl-2-(m -azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate (sAND), N-succinimidyl-4(4-azidophenyl) 1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)-1,3 'dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3'-dithiopropionate (sAED) , sulfosucci- 15 045580 7-azido-4-methylcumine-3-acetate (sulfo-sAMCA), p-nitrophenyldiazopyruvate (pNPDP), pnitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), 1 -(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(ρ-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, p-azidobenzoylhydrazide (AVH), 4-(p-azidosalicylamido)butylamine (AsBA) or pazidophenylglyoxal (APG). In some embodiments, the linker comprises a cleavable linker, optionally comprising a dipeptide linker. In some embodiments, the dipeptide linker comprises Val-Cit, Phe-Lys, Val-Ala, or Val-Lys. In some embodiments, the linker comprises a non-cleavable linker. In some embodiments, the linker contains a maleimide group, optionally including maleimidocaproyl (mc), succinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC ). In some embodiments, the linker further comprises a spacer. In some embodiments, the spacer comprises p-aminobenzyl alcohol (PAB), p-aminobenzyloxycarbonyl (PABC), a derivative or analog thereof. In some embodiments, the conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, the additional conjugating moiety is capable of extending the serum half-life of the IL-2 conjugate. In some embodiments, a form of IL-2 suitable for use in the present invention is a fragment of any of the forms of IL-2 described herein. In some embodiments, a form of IL-2 suitable for use in the present invention is pegylated, as described in US Patent Application Publication No. US 2020/0181220 A1 and US Patent Application Publication No. US 2020/0330601 A1. In some embodiments, a form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently attached to a conjugate moiety containing polyethylene glycol (PEG), wherein: the IL-2 polypeptide contains an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 5; and AzK replaces an amino acid at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with respect to the amino acid positions in SEQ ID NO: 5. In some embodiments, the IL-2 polypeptide contains N -terminal deletion of one residue relative to SEQ ID NO: 5. In some embodiments, a form of IL-2 suitable for use in the present invention has no interaction with the alpha chain of IL-2R, but retains normal binding to the beta-gamma signaling complex IL-2R with intermediate affinity. In some embodiments, a form of IL-2 suitable for use in the present invention is an IL-2 conjugate comprising: an IL-2 polypeptide containing N6azidoethoxy-L-lysine (AzK) covalently attached to a polyethylene glycol (PEG) containing conjugate moiety ), wherein: the IL-2 polypeptide contains an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 5; and AzK replaces an amino acid at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with respect to the amino acid positions in SEQ ID NO: 5. In some embodiments, a form of IL-2 suitable for use in the present invention, is an IL-2 conjugate comprising: an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently attached to a polyethylene glycol (PEG) containing conjugate moiety, wherein: the IL-2 polypeptide contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 5; and AzK replaces an amino acid at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69, or L72 with respect to the amino acid positions in SEQ ID NO: 5. In some embodiments, a form of IL-2 suitable for use in the present invention, is an IL-2 conjugate comprising: an IL-2 polypeptide containing N6-azidoethoxy-L-lysine (AzK) covalently attached to a polyethylene glycol (PEG) containing conjugate moiety, wherein: the IL-2 polypeptide contains an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 5; and AzK replaces an amino acid at positions K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 or L72 with respect to amino acid positions b SEQ ID NO: 5.

In some embodiments, a form of IL-2 suitable for use in the present invention is nemvaleukin alpha, also known as ALKS-4230 (SEQ ID NO: 6), which is available from Alkermes, Inc. Nemvaleukin alpha is also known as a fragment of human interleukin 2 (1-59), variant (Cys ¹²⁵ > Ser ⁵¹ ), fused through a peptidyl linker ( ⁶⁰ GG ⁶¹ ) with a fragment of human interleukin 2 (62-132), fused through a peptidyl linker ( ¹³³ GSGGGS ¹³⁸ ) with a human fragment of the interleukin 2 receptor α chain (139-303), produced by Chinese hamster ovary (CHO) cells, glycosylated; human interleukin 2 (IL-2) (75-133) peptide [Cys ¹²⁵ (51)>Ser] mutant (1-59) fused via peptide linker G2 (60-61) with human interleukin 2 (IL-2 ) (4-74)-peptide (62-132) and through the peptide linker GSG3S (133-138) with the α-chain of the human interleukin 2 receptor (alpha subunit IL2R, IL2Ra, IL2RA) (1-165)-peptide (139 -303), produced in Chinese hamster ovary (CHO) cells, alpha glycoform. The amino acid sequence of nemvaleukin alpha is set forth in SEQ ID NO: 6. In some embodiments, nemvaleukin

- 16 045580 kin alpha exhibits the following post-translational modifications: disulfide bridges at positions: 31-116, 141-285, 184-242, 269-301, 166-197 or 166-199, 168-199 or 168-197 (using numbering in SEQ ID NO: 6), and glycosylation sites at positions: N187, N206, T212 using numbering in SEQ ID NO: 6. Preparation and properties of nemvaleukin alpha, as well as additional alternative forms of IL-2 suitable for use in the present invention , are described in US Patent Application Publication No. US 2021/0038684 A1 and US Patent No. 10183979, the disclosure of which is incorporated herein by reference. In some embodiments, a form of IL-2 suitable for use in the present invention is a protein having at least 80%, at least 90%, at least 95%, or at least 90% sequence identity to SEQ ID NO : 6. In some embodiments, a form of IL-2 suitable for use in the present invention has the amino acid sequence set forth in SEQ ID NO: 6, or conservative amino acid substitutions thereof. In some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising amino acids 24-452 of SEQ ID NO: 7 or variants, fragments or derivatives thereof. In some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, or at least 90% identity amino acid sequence. 24-452 in SEQ ID NO: 7 or variants, fragments or derivatives thereof. Other forms of IL-2 suitable for use in the present invention are described in US Pat. No. 10183979, the disclosure of which is incorporated herein by reference. Optionally, in some embodiments, a form of IL-2 suitable for use in the present invention is a fusion protein comprising a first fusion partner that is linked to a second fusion partner by a mucin domain polypeptide linker, wherein the first fusion partner is IL-IRa or a protein having at least 98% amino acid sequence identity with IL-IRa and having IL-Ra receptor antagonist activity, and wherein the second fusion partner comprises all or a portion of an immunoglobulin containing an Fc region, wherein the mucin domain polypeptide linker comprises SEQ ID NO : 8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8, and wherein the half-life of the fusion protein is improved compared to fusion of the first fusion partner to the second fusion partner in the absence of a mucin domain polypeptide linker.

- 17 045580

table 2

Amino acid sequences of interleukins

Identifier Sequence (single letter amino acid symbols) SEQ Ш NO3 recombinant human IL-2 (rhIL-2) MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL 60 EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120 RWITFCQSII STLT 134 SEQ Ш NO:4 Aldesleykin PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE 60 ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120 ITFSQSIISTLT 132 SEQIDNO:5 form IL-2 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE 60 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120 WITFCQSIIS TLT 133 SEQ Ш NO:6 Nemvaleikin alpha SKNFHLRPRD LISNINVIVL ELKGSETTFM CEYADETATI VEFLNRWITF SQSIISTLTG 60 GSSSTKKTQL QLEHLLLDLQ MILNGINNYK NPKLTRMLTF KFYMPKKATE LKHLQCLEEE 120 LKPLEEVLNL AQGSGGGSEL CDDDPPEIPH ATFKAMAYKE GTMLNCECKR GFRRIKSGSL 180 YMLCTGNSSH SSWDNQCQCT SSATRNTTKQ VTPQPEEQKE RKTTEMQSPM QPVDQASLPG 240 HCREPPPWEN EATERIYHFV VGQMVYYQCV QGYRALHRGP AESVCKMTHGKTRWTQPQLI 300 CTG303 SEQ Ш NO:7 form IL-2 MDAMKRGLCC VLLLCGAVFV SARRPSGRKS SKMQAFRIWD VNQKTFYLRN NQL VAGYLQG 60 PNVNLEEKID VVPIEPHALF LGIHGGKMCL SCVKSGDETR LQLEAVNITD LSENRKQDKR 120

- 18 045580

FAFIRSDSGP TTSFESAACP GWFLCTAMEA DQPVSLTNMP DEGVMVTKFY FQEDESGSGG 180 ASSESSASSD GPHPVITESR ASSESSASSD GPHPVITESR EPKSSDKTHT CPPCPAPELL 240 GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ 300 YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR 360 EEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS 420 RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 452 SEQIDNO:8 polypeptide with mucin domain SESSASSDGP HPVITP 16 SEQ Ш NO:9 recombinant human IL-4 (rhIL-4) MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH 60 EKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120 MREKYSKCSS 130 SEQ Ш NO: 10 recombinant human IL-7 (rhIL-7) MDDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA 60 ARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120 KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH 153 SEQ Ш NO: And recombinant human IL-15 (rhIL-15) MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQVISLESGDASI 60 HDTVENLIIL ANNSLSSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINTS 115 SEQ ID NO: 12 recombinant human IL-21 (rhIL-21) MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG 60 NNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120 HLSSRTHGSEDS 132

In some embodiments, a form of IL-2 suitable for use in the present invention comprises an antibody-cytokine grafted protein comprising a heavy chain variable region () including the complementarity determining regions HCDR1, HCDR2, HCDR3; light chain variable region (V _L ), including LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into CDR V _H or VL, wherein the antibody-cytokine grafted protein preferentially expands effector T cells over regulatory T cells. In one embodiment, the antibody-cytokine grafted protein comprises a heavy chain variable region (VH) including the complementarity determining regions HCDR1, HCDR2, HCDR3; light chain variable region (V _L ), including LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into a VH or VL CDR, wherein the IL-2 molecule is a mutein, and wherein the antibody-cytokine grafted protein preferentially expands effector T cells over regulatory T cells.

- 19 045580 mi T cells. In one embodiment, the IL-2 regimen includes administration of an antibody described in US Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference. In one embodiment, the cytokine antibody-grafted protein comprises a heavy chain variable region (VH) including the complementarity determining regions HCDR1, HCDR2, HCDR3; light chain variable region (VL), including LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into a VH or VL CDR, wherein the IL-2 molecule is a mutein, wherein the antibody-cytokine grafted protein preferentially expands effector T cells over regulatory T cells, and wherein the antibody further contains an IgG heavy chain and an IgG light chain selected from the group consisting of: an IgG light chain containing SEQ ID NO: 39 and an IgG heavy chain containing SEQ ID NO: 38; an IgG light chain containing SEQ ID NO: 37 and an IgG heavy chain containing SEQ ID NO: 29; an IgG light chain containing SEQ ID NO: 39 and an IgG heavy chain containing SEQ ID NO: 29; and an IgG light chain comprising SEQ ID NO: 37 and an IgG heavy chain comprising SEQ ID NO: 38.

In one embodiment, an IL-2 molecule or fragment thereof is grafted into an HCDR1 _VH , wherein the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or fragment thereof is grafted into an HCDR2 _VH , wherein the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or fragment thereof is grafted into an HCDR3 _VH , wherein the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or fragment thereof is grafted into the LCDR1 VL, wherein the IL-2 molecule is a mutein. In one embodiment, the IL-2 molecule or fragment thereof is grafted into the LCDR2 VL, wherein the IL-2 molecule is a mutein. In one embodiment, an IL-2 molecule or fragment thereof is grafted into an LCDR3 VL, wherein the IL-2 molecule is a mutein.

The insertion of the IL-2 molecule may be in or near the N-terminal CDR region, in the middle CDR region, or in or near the C-terminal CDR region. In some embodiments, the antibody-cytokine grafted protein comprises an IL-2 molecule embedded in a CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In some embodiments, the antibody-cytokine grafted protein comprises an IL-2 molecule embedded in a CDR, wherein the IL-2 sequence completely or partially replaces the CDR sequence. The replacement with an IL-2 molecule may be at or near the N-terminal CDR region, the middle CDR region, or the C-terminal CDR region. The replacement with an IL-2 molecule may consist of just one or two amino acids in the CDR sequence or the entire CDR sequence.

In some embodiments, the IL-2 molecule is grafted directly into the CDR without a peptide linker, without additional amino acids between the CDR sequence and the IL-2 sequence. In some embodiments, the IL-2 molecule is indirectly linked to the CDR by a peptide linker with one or more additional amino acids between the CDR sequence and the IL-2 sequence.

In some embodiments, the IL-2 molecule described herein is an IL-2 mutein. In some cases, the IL-2 mutein contains the R67A substitution. In some embodiments, the IL-2 mutein contains the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the IL-2 mutein contains the amino acid sequence shown in table. 1 in US Patent Application Publication No. US 2020/0270334 A1, the disclosure of which is incorporated herein by reference.

In one embodiment, the antibody-cytokine grafted protein comprises HCDR1 selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, and SEQ ID NO: 25. In one embodiment, the grafted protein cytokine antibody comprises HCDR1 selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13 and SEQ ID NO: 16. In one embodiment, the cytokine antibody grafted protein comprises HCDR1 selected from the group consisting of HCDR2 selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 20, SEQ ID NO: 23 and SEQ ID NO: 26. In one embodiment, the antibody-cytokine grafted protein comprises HCDR3, selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24 and SEQ ID NO: 27. In one embodiment, the antibody-cytokine grafted protein comprises a V _H region containing the amino acid sequence of SEQ ID NO: 28. In one embodiment, the cytokine antibody-grafted protein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 29. In one embodiment, the cytokine antibody-grafted protein comprises a VL region comprising the amino acid sequence of SEQ ID NO: 36. In one embodiment In an embodiment, the cytokine antibody-grafted protein comprises a light chain containing the amino acid sequence of SEQ ID NO: 37. In one embodiment, the cytokine antibody-grafted protein comprises a VH region containing the amino acid sequence of SEQ ID NO: 28 and a VL region containing the amino acid sequence sequence SEQ ID NO: 36. In one embodiment, the antibody-cytokine grafted protein comprises a heavy chain region containing the amino acid sequence of SEQ ID NO: 29 and a light chain region containing

- 20 045580 containing the amino acid sequence of SEQ ID NO: 37. In one embodiment, the antibody-cytokine grafted protein comprises a heavy chain region containing the amino acid sequence of SEQ ID NO: 29 and a light chain region containing the amino acid sequence of SEQ ID NO: 39. In one embodiment, the antibody-cytokine-grafted protein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 38 and a light chain region comprising the amino acid sequence of SEQ ID NO: 37. In one embodiment, the antibody-cytokine-grafted protein comprises a heavy chain region chain containing the amino acid sequence of SEQ ID NO: 38, and a light chain region containing the amino acid sequence of SEQ ID NO: 39. In one embodiment, the antibody-cytokine grafted protein comprises IgG.IL2F71A.H1 or IgG.IL2R67A.H1 from the patent application publication US No. 2020/0270334 A1, or variants, derivatives or fragments thereof, or conservative amino acid substitutions thereof, or proteins with at least 80%, at least 90%, at least 95% or at least 98% sequence identity. In one embodiment, components of the antibody-cytokine grafted protein antibodies described herein include immunoglobulin sequences, framework sequences, or CDR sequences of palivizumab. In some embodiments, the antibody-cytokine grafted protein described herein has a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin or a comparable molecule. In one embodiment, the antibody-cytokine grafted protein described herein has the sequence set forth in Table. 3.

Table 3

Sequences of exemplary proteins grafted with plivizumab-IL-2 antibody

Identifier R Sequence (single letter amino acid symbols) SEQIDNO:13 IL-2 MYRMQLLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML 60 TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE 120 TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT 153 SEQ Ш NO: 14 mutein IL-2 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TFKFYMPKKA TELKHLQCLE 60 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE

- 21 045580

TTFMCEYADE TATIVEFLNR 120 WITFCQSIIS TLT 133 SEQ ID NO: 15 IL-2 mutein APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLE 60 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120 WITFCQSIIS TLT 133 SEQ ID NO: 16 HCDR1IL-2 GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTFKFYM PKKATELKHL 60 QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120 FLNRWITFCQ SIISTLTTSTS GMSVG 145 SEQ ID NO: 17 HCDR2 DIWWDDKKDY NPSLKS 16 SEQ ID NO: 18 HCDR3 SMITNWYFDV 10 SEQ ID NO: 19 HCDR1IL-2 no Kabat APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTAML TFKFYMPKKA TELKHLQCLE 60 EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR 120 WITFCQSIIS TLTSTSGMSV G 141 SEQ ID NO:20 HCDR2 no Kabat DIWWDDKKDY NPSLKS 16 SEQIDNO:21 HCDR3 no Kabat SMITNWYFDV 10 SEQ ID NO:22 HCDR1IL-2 no Clothia GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTFKFYM PKKATELKHL 60 QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120 FLNRWITFCQ SIISTLTTSTS GM 142 SEQ ID NO:23 HCDR2 no WWDDK 5

- 22 045580

Clothia SEQ ID NO:24 HCDR3 no Clothia SMITNWYFDV 10 SEQ ID NO:25 HCDR1IL-2 IMGT GFSLAPTSSS TKKTQLQLEH LLLDLQMILN GINNYKNPKL TAMLTFKFYM PKKATELKHL 60 QCLEEELKPL EEVLNLAQSK NFHLRPRDLI SNINVIVLEL KGSETTFMCE YADETATIVE 120 FLNRWITFCQ SIISTLTTSTS GMS 143 SEQ ID NO:26 HCDR2 IMGT IWWDDKK 7 SEQ ID NO:27 HCDR3 IMGT ARSMITNWYF DV 12 SEQ ID NO:28 _VH QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY 60 KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLRPRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSS 253 SEQ ID NO:29 Heavy chain QMILNGINNY KNPKLTAMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR 60 PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG 120 WIRQPPGKAL EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC 180 ARSMITNWYF DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV 240 TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR 300 VEPKSCDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK 360 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL

- 23 045580

NGKEYKCKVS NKALAAPIEK 420 TISKAKGQPR EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT 480 PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 533 SEQ Ш NO:30 LCDR1 no Kabat KAQLSVGYMH 10 SEQIDNO:31 LCDR2 no Kabat DTSKLAS 7 SEQ ID NO:32 LCDR3 no Kabat FQGSGYPFT 9 SEQ ID NO:33 LCDR1 no Chothia QLSVGY 6 SEQ ID NO:34 LCDR2 no Chothia DTS 3 SEQIDNO:35 LCDR3 no Chothia GSGYPF 6 SEQ ID NO:36 v _L DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIK 106 SEQ ID NO:37 Light chain DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213

- 24 045580

SEQIDNO:38 Light chain QVTLRESGPA LVKPTQTLTL TCTFSGFSLA PTSSSTKKTQ LQLEHLLLDL QMILNGINNY 60 KNPKLTRMLT AKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLRPRDLISNINV 120 IVLELKGSET TFMCEYADET ATIVEFLNRW ITFCQSIIST LTSTSGMSVG WIRQPPGKAL 180 EWLADIWWDD KKDYNPSLKS RLTISKDTSK NQVVLKVTNM DPADTATYYC ARSMITNWYF 240 DVWGAGTTVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT 300 SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKR VEPKSCDKTH 360 TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV AVSHEDPEVK FNWYVDGVEV 420 HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NI< ALA APIEK TISKAKGQPR 480 EPQVYTLPPS REEMTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF 540 FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 583 SEQ Ш NO:39 Light chain DIQMTQSPST LSASVGDRVT ITCKAQLSVG YMHWYQQKPG KAPKLLIYDT SKLASGVPSR 60 FSGSGSGTEF TLTISSLQPD DFATYYCFQG SGYPFTFGGG TKLEIKRTVA APSVFIFPPS 120 DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180 SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 213

The term IL-4 (also referred to herein as IL4) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and eosinophils, basophils and mast cells. IL-4 regulates the differentiation of naïve T helper cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Once activated by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression and induces class switching to IgE and IgG1 expression from B cells. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-211) and ThermoFisher Scientific, Inc. ., Waltham, Massachusetts, USA (Recombinant Human IL-15 Protein, Cat. No. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is presented in table. 2 (SEQ ID NO: 9).

The term IL-4 (also referred to herein as IL4) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and eosinophils, basophils and mast cells. IL-4 regulates the differentiation of naive T helper cells (Th0 cells) into Th2 T cells. Steinke and Borish, Respir. Res. 2001, 2, 66-70. Once activated by IL-4, Th2 T cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 also stimulates B cell proliferation and MHC class II expression and induces class switch expression of IgE and IgG ₁ from B cells. Recombinant human IL-4 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd.,

- 25 045580

East Brunswick, NJ, USA (cat. no. CYT-211) and ThermoFisher Scientific, Inc., Waltham, MA, USA (recombinant human IL-15 protein, cat. no. Gibco CTP0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is presented in table. 2 (SEQ ID NO: 5).

The term IL-7 (also referred to herein as IL7) refers to a tissue-derived glycosylated cytokine known as interleukin 7, which can be derived from stromal and epithelial cells, as well as dendritic cells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate T cell development. IL-7 binds to the IL-7 receptor, a heterodimer consisting of IL-7 receptor alpha and the common gamma chain receptor, which in a series of signals are important for T cell development in the thymus and survival in the periphery. Recombinant human IL-7 suitable for use in the present invention is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-254) and ThermoFisher Scientific, Inc. ., Waltham, Massachusetts, USA (Recombinant Human IL-15 Protein, Cat. No. Gibco PHC0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is presented in table. 2 (SEQ ID NO: 6).

The term IL-15 (also referred to herein as IL15) refers to the T cell growth factor known as interleukin-15 and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001, 97, 14-32, the description of which is incorporated herein by reference. IL-15 shares signaling receptor β and γ subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain containing 114 amino acids (and an N-terminal methionine) with a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham, USA. Massachusetts, USA (recombinant human IL-15 protein, cat. no. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is presented in table. 2 (SEQ ID NO: 7).

The term IL-21 (also referred to herein as IL21) refers to the pleiotropic cytokine protein known as interleukin-21 and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the description of which is incorporated herein by reference. IL-21 is mainly produced by natural killer T cells and activated human CD4+ T cells. Recombinant human IL-21 is a single non-glycosylated polypeptide chain containing 132 amino acids with a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from several suppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, NJ, USA (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., Waltham, Massachusetts, USA , USA (recombinant human protein IL-21, cat. no. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is presented in table. 2 (SEQ ID NO: 8).

When an antitumor effective amount, a tumor inhibitory effective amount, or a therapeutic amount is indicated, the exact amount of the composition of the present invention to be administered may be determined by the physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient ( subject). In general, it can be stated that a pharmaceutical composition containing tumor-infiltrating lymphocytes (eg, secondary TILs or genetically modified cytotoxic lymphocytes) described herein can be administered at a dose of 10 ⁴ to 10 ¹¹ cells/kg body weight (eg, 105 to 106, from 105 to 10 ¹⁰ , from 105 to 10 ¹¹ , from 106 to 10 ¹⁰ , from 106 to 10 ¹¹ , from 10 ⁷ to 10 ¹¹ , from 10 ⁷ to 10 ¹⁰ , from 108 to 10 ¹¹ , from 108 to 10 ¹⁰ , 10 ⁹ to 10 ¹¹ , or 10 ⁹ to 10 ¹⁰ cells/kg body weight), including all integer values in these ranges. Compositions of tumor-infiltrating lymphocytes (including in some cases genetically modified cytotoxic lymphocytes) can also be administered multiple times at these dosages. Tumor-infiltrating lymphocytes (including in some cases genetically modified cytotoxic lymphocytes) can be administered using infusion techniques that are widely known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988 ). A person skilled in the art of medicine can easily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient for signs of disease and adjusting treatment accordingly.

The term hematologic malignancy or terms of similar meaning refer to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including, but not limited to, blood, bone marrow, lymph nodes, and lymphatic system tissues. Hematologic malignancies are also called liquid tumors. Hematological malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma and non-Hodgkin's lymphomas. The term B-cell hematologic malignancy refers to hematologic malignancies that affect B cells.

The term solid tumor refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term solid malignant tumor refers to malignant, neoplastic or cancerous solid tumors. Solid malignancies include, but are not limited to, sarcomas, carcinomas, and lymphomas such as lung, breast, prostate, colon, rectal, and bladder cancers. The tissue structure of solid tumors includes interdependent tissue compartments, including parenchyma (cancer cells) and supporting stromal cells, in which cancer cells are dispersed and which can provide a supportive microenvironment.

The term liquid tumor refers to an abnormal mass of cells that is liquid in nature. Liquid cancers include, but are not limited to, leukemias, myelomas and lymphomas, as well as other hematologic malignancies. TILs derived from liquid tumors may also be referred to herein as bone marrow infiltrating lymphocytes (MILs). TILs derived from liquid tumors, including liquid tumors circulating in the peripheral blood, may also be referred to herein as PBL. The terms MIL, TIL and PBL are used interchangeably herein and differ only depending on the type of tissue from which the cells are derived.

The term microenvironment as used herein may refer to the microenvironment of a solid or hematologic tumor as a whole or to a specific subset of cells within the microenvironment. The tumor microenvironment, as used herein, refers to the complex mixture of cells, soluble factors, signaling molecules, extracellular matrices, and mechanical cues that promote neoplastic transformation, support tumor growth and invasion, protect the tumor from host immunity, promote therapeutic resistance, and provide niches for dominant tumor tumors to thrive. metastases, as described in Swartz, et al., Cancer Res., 2012, 72, 2473. Although tumors express antigens that must be recognized by T cells, tumor clearance by the immune system is rare due to immune suppression by the microenvironment.

The term dynamic scheduling as used herein may refer to the creation of a flexible schedule for events to take place based on the outcome or result of one or more subsequent events. Thus, the scheduled date of an event, such as a patient treatment event, can be dynamically changed based on the outcome or result of one or more production steps performed after scheduling the scheduled date but before the event.

In one embodiment, the invention provides a method of treating cancer using a population of TILs, wherein the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TILs of the present invention. In some embodiments, a population of TILs may be provided in which the patient is pretreated with non-myeloablative chemotherapy prior to infusion of the TILs of the present invention. In one embodiment, the nonmyeloablative chemotherapy is cyclophosphamide at a dose of 60 mg/kg/day for 2 days (days 27 and 26 before TIL infusion) and fludarabine at a dose of 25 mg/m ² /day for 5 days (days 27 to 23 before TIL infusion). In one embodiment, following non-myeloablative chemotherapy and TIL infusion (on day 0) of the present invention, the patient receives an intravenous infusion of IL-2 intravenously at a dose of 720,000 IU/kg every 8 hours until physiologically tolerated.

Experimental evidence suggests that lymphodepletion prior to adoptive transfer of tumor-specific T cells plays a key role in enhancing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (cytokine residues). Accordingly, in some embodiments, a lymphodepletion step (sometimes also referred to as immunosuppressive conditioning) is administered to the patient prior to administration of the rTIL of the present invention.

The terms co-administration, administered in combination with, administration in combination with, simultaneous and parallel as used herein cover the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the present invention, for example, at least one potassium channel agonist in combination with multiple TIL) in a subject such that both active pharmaceutical ingredients and/or their metabolites are present in the subject simultaneously. Co-administration includes simultaneous administration of separate compositions, administration of separate compositions at different times, or administration of a composition in which two or more active pharmaceutical ingredients are present. Preferred simultaneous administration from

- 27 045580 efficient compositions and the introduction of a composition in which both agents are present.

The term effective amount or therapeutically effective amount refers to that amount of a compound or combination of compounds as described herein that is sufficient to carry out the intended use, including, but not limited to, treatment of a disease. The therapeutically effective amount may vary depending on the intended use (in vitro or in vivo), the subject and disease state being treated (eg, weight, age and sex of the subject), severity of the disease state, or route of administration. The term also applies to the dose that will produce a specific response in target cells (eg, decreased platelet adhesion and/or cell migration). The specific dosage will vary depending on the particular compounds selected, the dosage regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the tissues into which it is administered, and the physical delivery system in which the compound is carried.

The terms treatment, treatment process, treat and the like refer to obtaining the desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms and/or may be therapeutic in terms of partially or completely curing a disease and/or an adverse event associated with that disease. As used herein, the term treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the onset of the disease in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibition of the disease, i.e. cessation of its development or progression; and (c) alleviation of the disease, i.e. regression of the disease and/or alleviation of one or more symptoms of the disease. Treatment also involves the delivery of an agent to provide a pharmacological effect even in the absence of a disease or condition. For example, treatment includes delivery of a composition that can induce an immune response or confer immunity in the absence of a disease state, such as in the case of vaccination.

The term heterologous, when applied to portions of a nucleic acid or protein, indicates that the nucleic acid or protein contains two or more subsequences that are not found in equal proportion to each other in nature. For example, a nucleic acid is typically produced recombinantly by having two or more sequences from unrelated genes arranged to produce a new functional nucleic acid, such as a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein indicates that the protein contains two or more subsequences that are not found in the same relationship to each other in nature (eg, a fusion protein).

As used herein, the terms sequence identity, percent identity, and percent sequence identity (or synonyms thereof, e.g., 99% identical) of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a certain percentage of nucleotides or amino acid residues that are the same, when compared and matched (gaps as necessary) for maximum consistency, without considering any conservative amino acid substitutions as part of sequence identity. Percent identity can be determined using sequence comparison software or algorithm, or by visual assessment. Various algorithms and software known in the art can be used to obtain amino acid or nucleotide sequence alignments. Suitable programs for determining percent sequence identity include, for example, the BLAST program suite available on the US Government National Center for Biotechnology Information BLAST website. Comparison of two sequences can be done using the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, South San Francisco, CA), or MegAlign, available from DNASTAR, are additional publicly available programs that can be used for sequence alignment. One skilled in the art can determine appropriate parameters for maximum matching using dedicated matching software. In some embodiments, default settings of the mapping software are applied.

As used herein, the term variant covers, but is not limited to, proteins, antibodies, or fusion proteins that contain an amino acid sequence that differs from the amino acid sequence of the reference protein, antibody, or fusion protein by one or more substitutions, deletions, and/or additions at specified positions within or adjacent to the amino acid sequence of a reference antibody, protein, or fusion protein. The variant may contain one or more conservative substitutions in its amino acid sequence compared to the amino acid sequence of the reference antibody. Conservative substitutions may include, for example,

- 28 045580 replacement of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody, protein, or fusion protein. The term variant also includes pegylated antibodies or proteins.

The term tumor-infiltrating lymphocytes or TILs as used herein refers to a population of cells originally derived as white blood cells that have left the subject's bloodstream and migrated into the tumor. TILs include, but are not limited to, cytotoxic CD8+ T cells (lymphocytes), Th1 and Th7 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include both primary and secondary TILs. Primary TILs are those TILs that are derived from patient tissue samples as described herein (sometimes referred to as freshly obtained or freshly isolated), and secondary TILs are any populations of TIL cells that have been expanded or proliferated as described herein, including , among other things, total TILs, propagated TILs (REP TILs), and reREP TILs as described herein. reREP TILs may include, for example, second propagation TILs or second additional propagation TILs (such as, for example, those described in step D of Fig. 2A and/or 9, including TILs referred to as reREP TILs).

TILs can usually be defined either biochemically, using cell surface markers, or functionally, by their ability to invade tumors and influence treatment. TILs can generally be classified by the expression of one or more of the following biomarkers: CD4, CD8, TCRae, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally determined to infiltrate solid tumors when reintroduced to a patient. TILs may be further characterized by activity - for example, TILs may be considered active if, for example, interferon (IFN) release is greater than about 50 pg/ml, greater than about 100 pg/ml, greater than about 150 pg/ml, or greater than about 200 pg/ml . TILs may be considered active if, for example, interferon (IFNy) release is greater than about 50 pg/ml, greater than about 100 pg/ml, greater than about 150 pg/ml, or greater than about 200 pg/ml, greater than about 300 pg/ml, greater than about 400 pg/ml, greater than about 500 pg/ml, greater than about 600 pg/ml, greater than about 700 pg/ml, greater than about 800 pg/ml, greater than about 900 pg/ml, greater than about 1000 pg/ml.

The terms pharmaceutically acceptable carrier or pharmaceutically acceptable excipient are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Unless any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, it is contemplated for use in the therapeutic compositions of the present invention. Additional active pharmaceutical ingredients, such as other drugs, may also be included in the described compositions and methods.

The terms about and approximately mean a value within a statistically significant range. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of the target value or range. The permissible variation covered by the terms about or approximate depends on the particular system being studied and can be readily estimated by one skilled in the art. Moreover, in the context of this document, the terms about and approximately mean that the dimensions, compositions, parameters, shapes and other quantities and characteristics do not have to be exact, but may be approximate and/or larger or smaller, as necessary, reflecting deviations, coefficients translation, rounding, measurement errors, etc., as well as other factors known to those skilled in the art. In general, size, composition, parameter, shape or other quantity or characteristic is described in terms of about or about, whether specifically stated or not. It should be noted that the described arrangements can be used in embodiments of a wide variety of sizes and shapes.

The transition terms containing, consisting essentially of, and consisting of, when used in the accompanying claims in their original and amended form, define the scope of the claims with respect to what unspecified additional claim elements or steps, if any, are excluded from the scope of the claims. The term comprising is intended to be inclusive or non-limiting and not to exclude any additional, unspecified elements, methods, steps or materials. The term consisting of excludes any element, step or material other than those specified in the claims, and, in the latter case, impurities typically associated with the specified material(s). The term essentially consists of limits the scope of the claims to those elements, steps or material(s), and those that do not substantially affect the essential and novel feature(s) of the claimed invention. All compositions, methods and kits described herein that embody the present invention, in alternative embodiments, may be more specifically defined by any of the transition terms comprising, consisting essentially of, and consisting of.

II. TIL production processes (variants of GEN3 processes).

Without being limited to any particular theory, it is believed that initiating a first expansion that initiates T cell activation, followed by a rapid second expansion that enhances T cell activation, as described in the methods of the present invention, produces expanded T cells that which retain a younger phenotype, and as such, the expanded T cells of the present invention are expected to exhibit greater cytotoxicity toward cancer cells than T cells expanded by other means. In particular, T cell activation is thought to be initiated by exposure to anti-CD3 antibody (e.g., OKT-3), IL-2, and optional antigen presenting cells (APCs) and then enhanced by subsequent exposure to additional anti-CD-3 antibody (e.g., OKT-3), IL-2 and APC, as specified in the methods of the present invention, limit or prevent the maturation of T cells in culture, yielding a population of T cells with a less mature phenotype, the T cells of which are less exhausted when expanded in culture and exhibit greater cytotoxicity against cancer cells. In some embodiments, the rapid second expansion step is divided into multiple steps to achieve increasing culture scaling by: (a) performing a rapid second expansion by culturing the T cells in a small scale culture in a first container, such as a GREX 100MCS container, for a period of about 3 to 4 days, and then (b) transferring the T cells in a small-scale culture into a second container larger than the first container, for example, a G-REX 500MCS container, and culturing the T cells from the small-scale culture in a larger culture in the second container for a period of about 4 to 7 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve down-scale culture by: (a) performing a rapid second expansion by culturing T cells in a first small-scale culture in a first container, e.g., a G-REX 100MCS container, for from about 3 to 4 days, and then (b) transfer and distribute T cells from the first small-scale culture to and among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 second containers equal in size to the first container, wherein in each second container a portion of the T cells from the first small-scale culture transferred to such second container are cultured in the second small-scale culture in over a period of about 4 to 7 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve down- and up-scale culture by: (a) performing a rapid second expansion by culturing T cells in a small scale culture in a first container, such as a G-REX 100MCS container, for period of about 3 to 4 days, and then (b) transferring and distributing T cells from the small-scale culture to and among at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19 or 20 second containers that are larger in size than the first container, for example G-REX 500MCS containers, each second container containing a portion of T cells from a small-scale culture transferred into such a second container, cultivated in a larger scale culture for a period of about 4 to 7 days. In some embodiments, the rapid expansion step is divided into multiple steps to achieve down- and up-scale culture by: (a) performing a rapid second expansion by culturing T cells in a small-scale culture in a first container, such as a G-REX 100MCS container, for about 4 days, and then (b) transferring and distributing the T cells from the small-scale culture into and among 2, 3 or 4 second containers that are larger in size than the first container, for example, G-REX 500MCS containers, each in a second container, a portion of the T cells from the small-scale culture transferred to the second container are cultured in the larger-scale culture for about 5 days.

An exemplary TIL process, known as Process 3 (also referred to herein as GEN3), containing some of these features is depicted in FIG. 2A and 2C and/or 9, and some of the advantages of this embodiment of the present invention compared to process 2A are described in FIG. 2B. Two embodiments of process 3 are shown in FIG. 2C. The 2A or Gen 2 process is also described in US Patent Publication No. 2018/.0280436, which is incorporated herein by reference in its entirety. Process 3 or Gen 3 is also described in International Patent Application No. PCT/US2019/059718, incorporated herein by reference in its entirety, and in FIG. 5, 6, 8, 9, 10 and 11 presented in this application.

In some embodiments, the rapid second expansion is performed after the T cell activation caused by the initiating first expansion begins to decline, weaken, attenuate, or decrease.

In some embodiments, the rapid second expansion is performed after the T cell activation caused by the initiating first expansion has decreased by exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,

32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

92, 93, 94, 95, 96, 97, 98, 99 or 100%.

In some embodiments, the rapid second expansion is performed after the T cell activation caused by the initiating first expansion has decreased by a percentage ranging from about 1% to 100%.

In some embodiments, rapid second expansion is performed after T cell activation caused by the initiating first expansion has decreased by a percentage in the range of about 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100%.

In some embodiments, the rapid second expansion is performed after the T cell activation caused by the initiating first expansion has decreased by at least exactly or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%.

In some embodiments, the rapid second expansion is performed after the T cell activation caused by the initiating first expansion has decreased by up to or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,

29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,

59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

In some embodiments, the decrease in T cell activation caused by the initiating first expansion is determined by a decrease in the amount of interferon gamma released by the T cells in response to antigen stimulation. Reduction in the amount of interferon gamma released by T cells by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 45%, 50%, 55%, 60% , 65%, 70% and/or 75% compared with baseline or control interferon gamma levels indicate decreased T cell activation. Reduction in the amount of interferon gamma released by T cells to less than 200 pg/ml, less than 250 pg/ml, less than 300 pg/ml, less than 350 pg/ml, less than 400 pg/ml, less than 450 pg/ml, less than 500 pg/ml, less than 550 pg/ml, less than 600 pg/ml, less than 650 pg/ml, less than 700 pg/ml, less than 750 pg/ml, less than 800 pg/ml, less than 850 pg/ml, less than 900 pg/ ml, less than 950 pg/ml or less than 1000 pg/ml indicates decreased T cell activation.

In some embodiments, the initial T cell expansion is performed over a period of up to or about 7 days or about 8 days.

In some embodiments, the initial T cell expansion is performed for a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

In some embodiments, the initial T cell expansion is performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days.

In some embodiments, rapid second expansion of T cells is performed over a period of up to or about 11 days.

In some embodiments, rapid second expansion of T cells is performed for a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days .

In some embodiments, rapid second T cell expansion is performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or 11 days.

In some embodiments, the triggering first T cell expansion is performed over a period of 1 day or so to 7 days or so, and the rapid second expansion of T cells is performed over a period of 1 day or so to 11 days or so.

In some embodiments, the initiating first T cell expansion is performed for a period of time up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days, and the rapid second T cell expansion carried out for a period of up to or about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or 11 days.

In some embodiments, the triggering first T cell expansion is performed over a period of 1 day or so to 8 days or so, and the rapid second expansion of T cells is performed over a period of 1 day or so to 9 days or so.

In some embodiments, the initial T cell expansion is performed for 8 days and the rapid second T cell expansion is performed for 9 days.

In some embodiments, the triggering first T cell expansion is performed over a period of 1 day or so to 7 days or so, and the rapid second expansion of T cells is performed over a period of 1 day or so to 9 days or so.

In some embodiments, the initial T cell expansion is performed in

- 31 045580 for 7 days, and rapid second expansion of T cells is performed for 9 days.

In some embodiments, the T cells are tumor infiltrating lymphocytes (TILs).

In some embodiments, the T cells are bone marrow infiltrating lymphocytes (MIL).

In some embodiments, the T cells are peripheral blood lymphocytes (PBLs).

In some embodiments, the T cells are obtained from a donor suffering from cancer.

In some embodiments, the T cells are TILs derived from a tumor excised from a patient suffering from cancer.

In some embodiments, the T cells are MIL derived from the bone marrow of a patient suffering from a hematologic malignancy.

In some embodiments, the T cells are PBL derived from peripheral blood mononuclear cells (PBMCs) from a donor. In some embodiments, the donor has cancer. In some embodiments, the cancer is a cancer selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer gland, human papillomavirus cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, human papillomavirus cancer, head and neck cancer ( including head and neck squamous cell carcinoma (HNSCC), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some embodiments, the donor suffers from a tumor. In some embodiments of the present invention, the tumor is a hematologic malignancy. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor suffers from a hematologic malignancy.

In some aspects of the present invention, immune effector cells, eg, T cells, can be obtained from a unit of blood collected from a subject using a variety of techniques known to one of ordinary skill in the art, such as a FICOLL unit. In one preferred aspect, cells from the circulating blood of an individual are obtained through apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, cells collected by apheresis may be washed to remove the plasma fraction and optionally place the cells in an appropriate buffer or medium for further processing steps. In one embodiment, the cells are washed with phosphate-buffered saline (PBS). In an alternative embodiment, the wash solution does not contain calcium and may not contain magnesium or may not contain many, if not all, divalent cations. In one aspect, T cells are isolated from peripheral blood lymphocytes by erythrocyte lysis and monocyte depletion, for example, by PERCOLL gradient centrifugation or countercurrent centrifugal elutriation.

In some embodiments, the T cells are PBL isolated from whole blood or an apheresis product enriched with donor lymphocytes. In some embodiments, the donor has cancer. In some embodiments, the cancer is a cancer selected from the group consisting of melanoma, ovarian cancer, endometrial cancer, thyroid cancer, colorectal cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer gland, human papillomavirus cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, human papillomavirus cancer, head and neck cancer ( including head and neck squamous cell carcinoma (HNSCC), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer and renal cell carcinoma. In some embodiments, the donor suffers from a tumor. In some embodiments of the present invention, the tumor is a hematologic malignancy. In some embodiments, the tumor is a solid tumor. In some embodiments, the donor suffers from a hematologic malignancy. In some embodiments, PBL is isolated from whole blood or lymphocyte-enriched apheresis product by positive or negative selection methods, i.e. by removing the PBL with marker(s), such as CD3 ⁺ CD45+, for a T cell phenotype, or by removing a cell of a non-T cell phenotype, leaving the PBL. In other embodiments, PBL is isolated by gradient centrifugation. Once PBL is isolated from donor tissue, the initiating first propagation of PBL can be initiated by plating

- 32 045580 suitable amount of allocated PBL (in some embodiments, approximately 1x10 ⁷

PBL) into the initiating first propagation culture in accordance with the initiating first propagation step of any of the methods described herein.

As discussed and generally described herein, TILs are taken from a patient sample and manipulated to expand their numbers prior to transplantation into the patient using the TIL expansion process described herein. In some embodiments, TILs may optionally be genetically manipulated, as discussed below. In some embodiments, TILs may be cryopreserved before or after expansion. Once thawed, they can also be re-stimulated to increase their metabolism before infusion into the patient.

In some embodiments, the initiating first propagation (including processes referred to herein as pre-rapid propagation (Pre-REP), as well as the processes shown in FIG. 2A as step B) is reduced to 1-8 days, and the rapid second propagation (including processes, referred to herein as the rapid propagation protocol (REP), as well as the processes shown in Fig. 2A as step D) is reduced to 1-9 days, as described in detail below, as well as in the examples and figures. In some embodiments, the initiating first propagation (including processes referred to herein as pre-rapid propagation (Pre-REP), as well as the processes shown in FIG. 2A as step B) is reduced to 1-8 days, and the rapid second propagation (including processes, referred to herein as the rapid propagation protocol (REP), as well as the processes shown in Fig. 2A as step D) is reduced to 1-8 days, as described in detail below, as well as in the examples and figures. In some embodiments, the initiating first propagation (including processes referred to herein as pre-rapid propagation (Pre-REP), as well as the processes shown in FIG. 2A as step B) is reduced to 1-7 days, and the rapid second propagation (including processes, referred to herein as the rapid propagation protocol (REP), as well as the processes shown in Fig. 2A as step D) is reduced to 1-9 days, as described in detail below, as well as in the examples and figures. In some embodiments, the initiating first propagation (including processes referred to herein as pre-rapid propagation (Pre-REP), as well as the processes shown in FIG. 2A as step B) is from 1 to 7 days, and the rapid second propagation (including processes referred to herein as the rapid propagation protocol (REP), as well as the processes shown in Fig. 2A as step D) ranges from 1 to 10 days, as described in detail below, as well as in the examples and figures. In some embodiments, the initiating first propagation (e.g., propagation described as step B in Fig. 2A) is reduced to 8 days, and the rapid second propagation (e.g., propagation as described in step D in Fig. 2A) is 7 to 9 days days. In some embodiments, the initiating first propagation (e.g., propagation described as step B in Fig. 2A) is 8 days, and the rapid second propagation (e.g., propagation as described in step D in Fig. 2A) is 8 to 9 days . In some embodiments, the initiating first propagation (e.g., propagation described as step B in Fig. 2A) is reduced to 7 days, and the rapid second propagation (e.g., propagation as described in step D in Fig. 2A) is 7 to 8 days days. In some embodiments, the initiating first propagation (eg, propagation described as step B in Fig. 2A) is reduced to 8 days, and the rapid second propagation (eg, propagation as described in step D in Fig. 2A) is 8 days. In some embodiments, the initiating first propagation (eg, propagation described as step B in FIG. 2A) is 8 days, and the rapid second propagation (eg, propagation as described in step D in FIG. 2A) is 9 days. In some embodiments, the initiating first propagation (eg, propagation described as step B in FIG. 2A) is 8 days, and the rapid second propagation (eg, propagation as described in step D in FIG. 2A) is 10 days. In some embodiments, the initiating first propagation (e.g., propagation described as step B in Fig. 2A) is 7 days, and the rapid second propagation (e.g., propagation as described in step D in Fig. 2A) is 7 to 10 days . In some embodiments, the initiating first propagation (e.g., propagation described as step B in Fig. 2A) is 7 days, and the rapid second propagation (e.g., propagation as described in step D in Fig. 2A) is 8 to 10 days . In some embodiments, the initiating first propagation (e.g., propagation described as step B in Fig. 2A) is 7 days, and the rapid second propagation (e.g., propagation as described in step D in Fig. 2A) is 9 to 10 days . In some embodiments, the initiating first breeding (eg, breeding described as step B in Fig. 2A) is reduced to 7 days, and the rapid second breeding (eg, breeding as described in step D in Fig. 2A) is from 7 to 9 days. days. In some embodiments, the combination of an initiating first propagation and a rapid second propagation (eg, propagations described as step B and step D in Fig. 2A) is 14-16 days, as detailed below and in the examples and figures. In particular, it is believed that some embodiments of the present invention include an initiating first propagation step in which TILs are activated by exposure to an anti-CD3 antibody, such as OKT-3, in the presence of IL-2 or exposure to an antigen in the presence of at least IL-2 and anti-CD3 antibodies, such as OKT-3. In some embodiments, the TILs that are activated during the initiating first propagation step, as described above, represent the first population of TILs, i.e. represent a population of primary cells.

In some embodiments, the TILs are not retained from the first propagation until the second propagation, but rather the TILs proceed directly to the second propagation (eg, in some embodiments, there is no persistence from step B to step D as shown in FIG. 2A). In some embodiments, the transition occurs in a closed system, as described herein. In some embodiments, TILs from the first propagation, the second population of TILs, move directly to the second propagation without a transition period.

In some embodiments, the first propagation, such as step B of FIG. 2A is performed in a closed system bioreactor. In some embodiments, a closed system is used to propagate TILs, as described herein. In some embodiments, a separate bioreactor is used. In some embodiments, the individual bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a separate bioreactor.

Designations A, B, C, etc. The steps specified herein refer to the non-limiting example of FIG. 2A and certain non-limiting embodiments described herein. The order of steps is below and in FIG. 2A is illustrative, and any combination or order of steps, as well as additional steps, repetition of steps, and/or omission of steps, are contemplated by this application and the methods described herein.

A. Cell viability assay.

Cell viability testing can be performed after initial first expansion (sometimes called initial expansion) using standard assays known in the art. Thus, in some embodiments, the method includes performing a cell viability assay after the initial first expansion. In some embodiments, a cell viability assay may be performed after a second expansion (eg, after REP) as well as after final collection. For example, a trypan blue assay can be performed on a sample of total TILs, which selectively labels dead cells and allows for assessment of viability. Other assays for viability testing may include, but are not limited to, the Alamar Blue assay and the MTT assay.

1. Cell count, viability, flow cytometry.

In some embodiments, cell number and/or viability are measured. The expression of markers, such as, but not limited to, CD3, CD4, CD8 and CD56, as well as any others disclosed or described herein, can be measured by flow cytometry with antibodies, such as, but not limited to, those commercially available from from BD Biosciences (BD Biosciences, San Jose, CA) using a FACSCanto™ flow pitometer (BD Biosciences). Cells can be counted manually using a disposable C-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed by any method known in the art, including, but not limited to, trypan blue staining. Cell viability can also be analyzed based on USSN 15/863634, which is incorporated herein by reference in its entirety. Cell viability can also be analyzed based on US Patent Publication No. 2018/0280436 or International Patent Publication No. WO/2018/081473, both of which are incorporated herein in their entirety for all purposes.

In some cases, the total TIL population can be cryopreserved immediately using the protocols described below. Alternatively, the total TIL population can be REPed and then cryopreserved as described below. Likewise, in the event that genetically modified TILs are to be used in a therapy, populations of total or REP TILs can be genetically modified for appropriate treatment.

III. TIL production processes (process options 2A).

An exemplary TIL process, known as process 2A, incorporating some of these features is depicted in FIG. 5, and some differences and advantages of this embodiment of the present invention compared to Process 1C are described in FIG. 6, and also in FIG. 11. Process 1C is shown for comparison in FIG. 6, 7 and 10. An embodiment of Process 2A is shown in FIG. 6, and also in FIG. 5, 9, 10 and 11. In FIG. 10 and 11 further illustrate exemplary process 2A compared to exemplary process 1C.

As described herein, the present invention may include the step of re-stimulating cryopreserved TILs to increase their metabolic activity and therefore relative health prior to transplantation into a patient, as well as methods for testing said metabolic health. As generally described herein, TILs are typically taken from a patient sample and manipulated to increase their number prior to transplantation into the patient. In some embodiments, TILs may optionally be genetically manipulated, as discussed below.

In some embodiments, TILs may be cryopreserved. Once thawed, they can also be re-stimulated to increase their metabolism before infusion into the patient.

In some embodiments, the first propagation (including processes called preREP, as well as the processes shown in FIG. 9 as step A) is reduced to 3-14 days, and the second propagation (including processes called REP, as well as the processes shown in FIG. 9 as stage B), is reduced to 7-14 days, as detailed below and in the examples and figures. In some embodiments, the first propagation (e.g., propagation described as step B in FIG. 9) is reduced to 11 days, and the second propagation (e.g., propagation as described in step D of FIG. 9) is reduced to 11 days, as described in the examples and shown in Fig. 5, 6, 8, 9, 10 and 11. In some embodiments, the combination of the first propagation and the second propagation (e.g., the propagations described as step B and step D in FIG. 9) is reduced to 22 days, as detailed below, and also in the examples and figures.

Designations A, B, C, etc. The steps below refer to FIGS. 9 and certain embodiments described herein. The order of steps is below and in Fig. 9 is illustrative, and any combination or order of steps, as well as additional steps, repetition of steps, and/or omission of steps, are contemplated by this application and the methods described herein.

A. Step A. Obtaining a sample of the patient's tumor.

In general, TILs are initially obtained from a patient tumor sample and then expanded to a larger population for further manipulation as described herein, optionally cryopreserved, restimulated as described herein, and optionally assessed for phenotype and metabolic parameters as an indicator of health TIL.

A sample of a patient's tumor can be obtained by methods known in the art, typically through surgical resection, core biopsy, core needle biopsy, core biopsy, or other methods of obtaining a sample that contains a mixture of tumor and TIL cells. In some embodiments, sampling from multiple lesions is employed. In some embodiments, surgical resection, core biopsy, core needle biopsy, core biopsy, or other methods of obtaining a sample that contains a mixture of tumor and TIL cells involves sampling from multiple lesions (i.e., sampling from one or more sites and/or or sites of tumor in a patient, as well as from one or more tumors in the same location or in close proximity). In general, a tumor sample can be taken from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor derived from a hematologic malignancy. A solid tumor may be from lung tissue. In some embodiments, the beneficial TILs are derived from non-small cell lung carcinoma (NSCLC).

Once obtained, the tumor sample is typically fragmented by sharp dissection into small pieces with a volume of from 1 to about 8 mm ³ , with pieces of about 2-3 mm ³ being particularly suitable. In some embodiments, TILs are cultured from these fragments using enzymatic tumor digestions. Such tumor digests can be prepared by incubation in an enzymatic medium (eg, Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate, 10 μg/ml gentamicin, 30 units/ml DNase, and 1.0 mg/ml collagenase) with subsequent mechanical dissociation (for example, using a tissue dissociator). Tumor digestion can be achieved by placing the tumor in an enzymatic medium and mechanically dissociating the tumor for approximately 1 min followed by incubation for 30 min at 37°C in 5% CO ₂ followed by repeated cycles of mechanical dissociation and incubation under the above conditions until no only small pieces of fabric. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using FICOLL branched hydrophilic polysaccharide to remove these cells. Alternative methods known in the art may be used, such as those described in US Patent Application Publication No. 2012/0244133 A1, the disclosure of which is incorporated herein by reference. Any of the above methods can be used in any of the embodiments described herein for TIL propagation methods or cancer treatment methods.

Tumor dissociating enzyme mixtures may include one or more dissociating enzymes such as, but not limited to, collagenase (including any mixture or type of collagenase), Accutase™, Accumax™, hyaluronidase, neutral protease (dispase), chymotrypsin, chymopapain , trypsin, caseinase, elastase, papain, type XIV protease (pronase), deoxyribonuclease I (DNase), trypsin inhibitor, any other dissociating or proteolytic enzyme, and any combination thereof.

In some embodiments, the dissociating enzymes are recovered from the lyophilized enzymes. In some embodiments, the lyophilized enzymes are reconstituted in a quantity of sterile buffer, such as HBSS.

In some cases, collagenase (eg, non-animal collagenase type 1) is reconstituted in 10 ml of sterile HBSS or other buffer. The lyophilized parent enzyme may have a concentration of 2892 units. PZ/bottle. In some embodiments, collagenase is reconstituted in 5 to 15 mL of buffer. In some embodiments, after reconstitution, the concentration of the collagenase solution ranges from about 100 units. PZ/ml up to about 400 units. PZ/ml, for example, from about 100 units. PZ/ml up to about 400 units. PZ/ml, from about 100 units. PZ/ml up to about 350 units. PZ/ml, from about 100 units. PZ/ml up to about 300 units. PZ/ml, from about 150 units. PZ/ml up to about 400 units. PZ/ml, about 100 units. PZ/ml, about 150 units. PZ/ml, about 200 units. PZ/ml, about 210 units. PZ/ml, about 220 units. PZ/ml, about 230 units. PZ/ml, about 240 units. PZ/ml, about 250 units. PZ/ml, about 260 units. PZ/ml, about 270 units. PZ/ml, about 280 units. PZ/ml, about 289.2 units. PZ/ml, about 300 units. PZ/ml, about 350 units. PZ/ml or about 400 units. PZ/ml.

In some embodiments, the neutral protease is reconstituted in 1 ml of sterile HBSS or other buffer. The lyophilized parent enzyme may have a concentration of 175 units. DMC/bottle. In some embodiments, after reconstitution, the concentration of the neutral protease solution is in the range of about 100 DMC/ml to about 400 DMC/ml, for example, from about 100 DMC/ml to about 400 DMC/ml, from about 100 DMC/ml to about 350 DMC/ml, from about 100 DMC/ml to about 300 DMC/ml, from about 150 DMC/ml to about 400 DMC/ml, about 100 DMC/ml, about 110 DMC/ml, about 120 DMC/ml, about 130 DMC/ml, about 140 DMC/ml, about 150 DMC/ml, about 160 DMC/ml, about 170 DMC/ml, about 175 DMC/ml, about 180 DMC/ml, about 190 DMC/ml, about 200 DMC/ ml, about 250 DMC/ml, about 300 DMC/ml, about 350 DMC/ml or about 400 DMC/ml.

In some embodiments, DNase I is reconstituted in 1 ml of sterile HBSS or other buffer. The lyophilized starting enzyme had a concentration of 4 KU/vial. In some embodiments, after reconstitution, the concentration of the DNase I solution is in the range of about 1 KU/ml to 10 KU/ml, for example, about 1 KU/ml, about 2 KU/ml, about 3 KU/ml, about 4 KU/ml , about 5 KU/ml, about 6 KU/ml, about 7 KU/ml, about 8 KU/ml, about 9 KU/ml or about 10 KU/ml.

In some embodiments, the enzyme solution is variable and its concentration may need to be determined. In some embodiments, the concentration of the lyophilized solution can be checked. In some embodiments, the final amount of enzyme added to the digestion mixture is adjusted based on the determined concentration of the solution.

In some embodiments, the enzyme mixture includes about 10.2 μl of neutral protease (0.36 U DMC/ml), 21.3 μl of collagenase (1.2 PZ/ml), and 250 μl of DNase I (200 U/ml) per volume of approximately 4.7 ml of sterile HBSS.

As stated above, in some embodiments, TILs are derived from solid tumors. In some embodiments, solid tumors are not fragmented. In some embodiments, solid tumors are not fragmented and are enzymatically digested as whole tumors. In some embodiments, tumors are digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase. In some embodiments, tumors are digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours. In some embodiments, tumors are digested in an enzyme mixture containing collagenase, DNase, and hyaluronidase for 1-2 hours at 37°C C, 5% CO ₂ . In some embodiments, tumors are digested in an enzyme mixture containing collagenase, DNase and hyaluronidase for 1-2 hours at 37°C, 5% CO ₂ with rotation. In some embodiments, tumors are digested overnight with constant rotation. In some embodiments, tumors are digested overnight at 37°C, 5% CO2 with constant rotation. In some embodiments, the entire tumor is combined with enzymes to form a tumor digestion reaction mixture.

In some embodiments, the tumor is reconstituted with lyophilized enzymes in a sterile buffer. In some embodiments, the buffer is sterile HBSS.

In some embodiments, the enzyme mixture contains collagenase. In some embodiments, the collagenase is collagenase IV. In some embodiments, the working solution for collagenase is a 10x working solution of 100 mg/ml.

In some embodiments, the enzyme mixture contains DNase. In some embodiments, the DNase working solution is a 10x working solution of 10,000 IU/mL.

In some embodiments, the enzyme mixture contains hyaluronidase. In some embodiments, the working solution for hyaluronidase is a 10x working solution of 10 mg/ml.

In some embodiments, the enzyme mixture contains 10 mg/ml collagenase, 1000

- 36 045580

IU/ml DNase and 1 mg/ml hyaluronidase.

In some embodiments, the enzyme mixture contains 10 mg/ml collagenase, 500

IU/ml DNase and 1 mg/ml hyaluronidase.

Typically, the collected cell suspension is called a primary cell population or a fresh cell population.

In some embodiments, fragmentation includes physical fragmentation, including, for example, cutting as well as splitting. In some embodiments, the fragmentation is physical fragmentation. In some embodiments, the fragmentation is a dissection. In some embodiments, fragmentation is accomplished through cleavage. In some embodiments, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients. In one embodiment, TILs can be initially cultured from enzymatic tumor digests and tumor fragments obtained from patients.

In some embodiments, when the tumor is a solid tumor, the tumor undergoes physical fragmentation after obtaining a tumor sample, for example, in stage A (as shown in Fig. 1). In some embodiments, fragmentation occurs prior to cryopreservation. In some embodiments, fragmentation occurs after cryopreservation. In some embodiments, fragmentation occurs after tumor acquisition and in the absence of any cryopreservation. In some embodiments, the tumor is fragmented and 10, 20, 30, 40 or more fragments or pieces are placed in each container for first expansion. In some embodiments, the tumor is fragmented and 30 or 40 fragments or pieces are placed in each container for first expansion. In some embodiments, the tumor is fragmented and 40 fragments or pieces are placed in each container for first expansion. In some embodiments, the multiple fragments include from about 4 to about 50 fragments, with each fragment having a volume of about 27 mm ³ . In some embodiments, the multiple fragments contain from about 30 to about 60 fragments with a total volume of from about 1300 mm ³ to about 1500 mm ³ . In some embodiments, the multiple fragments contain about 50 fragments with a total volume of about 1350 mm ³ . In some embodiments, the multiple fragments contain about 50 fragments with a total weight of from about 1 gram to about 1.5 grams. In some embodiments, the multiple fragments contain about 4 fragments.

In some embodiments, TILs are derived from tumor fragments. In some embodiments, the tumor fragment is obtained through sharp dissection. In some embodiments, the tumor fragment is from about 1 mm ³ to 10 mm ³ . In some embodiments, the tumor fragment is from about 1 mm ³ to 8 mm ³ . In some embodiments, the tumor fragment is about 1 mm ³ . In some embodiments, the tumor fragment is about 2 mm ³ . In some embodiments, the tumor fragment is about 3 mm ³ . In some embodiments, the tumor fragment is about 4 mm ³ . In some embodiments, the tumor fragment is about 5 mm ³ . In some embodiments, the tumor fragment is about 6 mm ³ . In some embodiments, the tumor fragment is about 7 mm ³ . In some embodiments, the tumor fragment is about 8 mm ³ . In some embodiments, the tumor fragment is about 9 mm ³ . In some embodiments, the tumor fragment is about 10 mm ³ . In some embodiments, the tumors measure 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumors measure 1 mm x 1 mm x 1 mm. In some embodiments, the tumors measure 2 mm x 2 mm x 2 mm. In some embodiments, the tumors measure 3 mm x 3 mm x 3 mm. In some embodiments, the tumors measure 4 mm x 4 mm x 4 mm.

In some embodiments, tumors are resected to minimize the amount of hemorrhagic, necrotic, and/or fatty tissue on each piece. In some embodiments, tumors are resected to minimize the amount of hemorrhagic tissue on each piece. In some embodiments, tumors are resected to minimize the amount of necrotic tissue on each piece. In some embodiments, tumors are resected to minimize the amount of fatty tissue in each piece.

In some embodiments, tumor fragmentation is performed to preserve the internal structure of the tumor. In some embodiments, tumor fragmentation is performed without performing a sawing motion with a scalpel. In some embodiments, TILs are derived from tumor digests. In some embodiments, tumor digests are prepared by incubation in an enzymatic medium, such as, but not limited to, RPMI 1640, 2 mM GlutaMAX, 10 mg/mL gentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in the enzymatic medium, the tumor can be mechanically dissociated within approximately 1 minute. The solution can then be incubated for 30 minutes at 37°C in 5% CO ₂ and then mechanically disrupted again for approximately 1 minute. After repeated incubation for 30 minutes at 37°C in 5% CO2, the tumor can be mechanically disrupted a third time in approximately 1 minute. In some embodiments, after the third mechanical dissociation, if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample with or without an additional 30 minutes of incubation at 37°C in 5% CO ₂ . In some embodiments, at the end of the last incubation, if the cell suspension contained a large number of red blood cells or dead cells, density gradient separation using Ficoll can be performed to remove these cells.

In some embodiments, the collected cell suspension prior to the first stage of expansion is referred to as a primary cell population or a freshly harvested cell population.

In some embodiments, the cells may optionally be frozen after sample collection and kept frozen until expansion described in Step B, which is described in more detail below and also illustrated in FIG. 9.

1. T cells and TILs from pleural exudate.

In some embodiments, the sample is a pleural fluid sample. In some embodiments, the source of T cells or TILs for expansion in accordance with the methods described herein is a pleural fluid sample. In some embodiments, the sample is a sample obtained from pleural exudate. In some embodiments, the source of T cells or TILs for expansion in accordance with the methods described herein is a sample obtained from pleural exudate, see, for example, the methods described in US patent publication US 2014/0295426, incorporated herein in its entirety. by reference for all purposes.

In some embodiments, any pleural fluid or pleural exudate suspected of and/or containing TILs can be used. Such a sample may be obtained from a primary or metastatic lung cancer such as NSCLC or SCLC. In some embodiments, the sample may be secondary metastatic cancer cells originating from another organ, such as breast, ovary, colon, or prostate. In some embodiments, the sample for use in the propagation methods described herein is pleural exudate. In some embodiments, the sample for use in the propagation methods described herein is a pleural transudate. Other biological samples may include other serous fluids containing TILs, including, for example, ascitic fluid from the abdominal cavity or fluid from a pancreatic cyst. Ascitic fluid and pleural fluid contain very similar chemical systems; both the abdominal cavity and the lung have mesothelial lines and fluid forms in the pleural cavity and peritoneal cavity in the same substance in malignancies, and such fluids in some embodiments contain TILs. In some embodiments where pleural fluid is exemplified herein, the same methods can be performed with ascites or other cystic fluids containing TILs with similar results.

In some embodiments, the pleural fluid is in its raw form, directly removed from the patient's body. In some embodiments, the raw pleural fluid is placed in a standard blood collection tube, such as an EDTA or heparin tube, prior to the contacting step. In some embodiments, the raw pleural fluid is placed in a standard Cell Save® tube (Veridex) prior to the contacting step. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient to avoid depletion of viable TILs. The number of viable TILs can be significantly reduced within 24 hours if left in untreated pleural fluid even at 4°C. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after collection from the patient. In some embodiments, the sample is placed in an appropriate collection tube within 1 hour, 5 hours, 10 hours, 15 hours, or up to 24 hours after collection from the patient at 4°C.

In some embodiments, the pleural fluid sample from the selected subject may be diluted. In one embodiment, the dilution is 1:10 pleural fluid to diluent. In another embodiment, the dilution is 1:9 pleural fluid to diluent. In another embodiment, the dilution is 1:8 pleural fluid to diluent. In another embodiment, the dilution is 1:5 pleural fluid to diluent. In another embodiment, the dilution is 1:2 pleural fluid to diluent. In another embodiment, the dilution is 1:1 pleural fluid to diluent. In some embodiments, diluents include saline, phosphate-buffered saline, another buffer, or a physiologically acceptable diluent. In some embodiments, the sample is placed in a CellSave tube immediately after collection from the patient and diluted to avoid the decline in viable TIL that can occur significantly within 24-48 hours if left in untreated pleural fluid, even at 4°C . In some embodiments, the pleural fluid sample is placed in an appropriate collection tube for 1 hour, 5 hours, 10 hours, 15 hours, 24 hours, 36 hours, up to 48 hours after collection from the patient. In some embodiments, the pleural fluid sample is placed in an appropriate collection tube for 1 hour, 5 hours, hours, 15 hours, 24 hours, 36 hours, up to 48 hours after collection from the patient and diluted at 4°C.

In yet another embodiment, pleural fluid samples are concentrated using conventional methods prior to further processing steps. In some embodiments, this pretreatment of the pleural fluid is preferred in circumstances where the pleural fluid needs to be cryopreserved for shipment to a laboratory performing the method or for subsequent analysis (eg, later than 24-48 hours after collection). In some embodiments, a pleural fluid sample is prepared by centrifuging the pleural fluid sample after it has been collected from the subject and resuspending the centrifugate or pellet in a buffer. In some embodiments, the pleural fluid sample is subjected to multiple centrifugation and resuspension prior to cryopreservation for transport or subsequent analysis and/or processing.

In some embodiments, pleural fluid samples are concentrated prior to further processing steps using a filtration method. In some embodiments, the pleural fluid sample used in the contacting step is prepared by filtering the fluid through a filter containing pores of known and substantially uniform size that allows the pleural fluid to pass through the membrane but retains tumor cells. In some embodiments, the membrane pore diameter may be at least 4 microns. In another embodiment, the pore diameter may be 5 μm or greater, and in another embodiment, any of 6, 7, 8, 9, or 10 μM. After filtration, cells, including TILs, retained by the membrane can be washed off the membrane into a suitable physiologically acceptable buffer. Cells, including TILs, concentrated in this manner can then be used in the contacting step of the process.

In some embodiments, a sample of pleural fluid (including, for example, untreated pleural fluid), diluted pleural fluid, or resuspended cell pellet is contacted with a lytic reagent that selectively lyses anucleate red blood cells present in the sample. In some embodiments, this step is performed prior to further processing steps under conditions where the pleural fluid contains a significant amount of red blood cells (RBC). Suitable lysis reagents include a single lytic reagent, or a lytic reagent and a quenching reagent, or a lytic reagent, a quenching reagent, and a fixation reagent. Suitable lytic systems are commercially available and include the BD Pharm Lyse™ system (Becton Dickenson). Other lytic systems include the Versalyse™ system, the FACSlyse™ system (Becton Dickenson), the Immunoprep™ system or the Erythrolyse II system (Beckman Coulter, Inc.) or the ammonium chloride system. In some embodiments, the lytic reagent may vary, with the main requirements being efficient lysis of red blood cells and preservation of the TILs and the phenotypic properties of the TILs in the pleural fluid. In addition to the use of a single lysis reagent, lytic systems useful in the methods described herein may include a second reagent, for example one that quenches or slows down the action of the lytic reagent in the remaining steps of the method, such as Stabilyse™ reagent. (Beckman Coulter, Inc.). A conventional fixing reagent may also be used depending on the choice of lytic reagents or the preferred implementation of the method.

In some embodiments, a pleural fluid sample, unprocessed, diluted, or repeatedly centrifuged or processed as described hereinabove, is cryopreserved at about -140° C. prior to further processing and/or expansion as provided herein.

B. Stage B. First reproduction.

1. Young TIL.

In some embodiments, the present methods provide young TILs that are capable of increasing replication cycles when administered to a subject/patient and, as such, may provide additional therapeutic benefits over older TILs (i.e., TILs that have subsequently undergone more number of replication cycles prior to administration to subject/patient). Signs of young TILs have been described in the literature, for example, Donia, at al., Scandinavian Journal of Immunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res, 16:6122–6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005); Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., J Immunother 32:415-423 (2009); Robbins, et al., J Immunol 2004; 173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, et al., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother, 31:742751 (2008), all of which are incorporated herein by reference in their entirety.

A variety of antigen receptors of T and B lymphocytes are formed as a result of somatic recombination of a limited but large number of gene segments. These gene segments include: V (variability), D (diversity), J (attachment) and C (constant) determine the binding specificity and subsequent applications of immunoglobulins and T-cell receptors (TCRs). The present invention provides a method for generating TILs that exhibit and enhance T cell repertoire diversity. In some embodiments, TILs produced by the present method exhibit an increase in T cell repertoire diversity. In some embodiments, TILs produced by the present method exhibit an increase in T cell repertoire diversity compared to freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs produced by the present method exhibit increased T cell repertoire diversity compared to fresh TILs and/or TILs produced by methods referred to as the 1C process, as presented in FIG. 10. In some embodiments, the TILs obtained from the first expansion exhibit an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is present in the immunoglobulin heavy chain. In some embodiments, the immunoglobulin variety is present in the immunoglobulin light chain. In some embodiments, diversity is present in the T cell receptor. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, an increase in T cell receptor (TCR) alpha and/or beta expression is observed. In some embodiments, an increase in T cell receptor (TCR) alpha expression is observed. In some embodiments, an increase in T cell receptor (TCR) beta expression is observed. In some embodiments, an increase in TCRab (ie, TCRa/β) expression is observed.

After cutting or splitting the tumor fragments, for example, as described in step A in FIG. 9, the resulting cells are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 ml wells in medium containing inactivated human AB serum with 6000 IU/ml IL-2. This population of primary cells is cultured for several days, typically 3 to 14 days, resulting in a population of total TILs, typically about 1x10 ^s total TILs. In some embodiments, this population of primary cells is cultured for a period of 7 to 14 days, resulting in a population of total TILs, typically about 1x10 ^s total TILs. In some embodiments, this population of primary cells is cultured for a period of 10 to 14 days, resulting in a population of total TILs, typically about 1x10 ^s total TILs. In some embodiments, this population of primary cells is cultured for about 11 days, resulting in a population of total TILs, typically about 1 x 10 ^s total TILs.

In a preferred embodiment, TIL propagation can be accomplished using an initial total TIL propagation step (e.g., described in step B of FIG. 9, which may involve processes called pre-REP) as described below and herein, followed by a second propagation (Stage D, including processes referred to as Rapid Expansion Protocol (REP) steps), as described below in Phase D and herein, followed by optional cryopreservation and subsequent second Step D (including processes referred to as REP re-stimulation steps), as described below and throughout this document. TILs resulting from this process may optionally be characterized by phenotypic characteristics and metabolic parameters as described herein.

In embodiments in which TIL cultures are initiated in 24-well plates, for example, using a Costar 24-well flat-bottom cell culture cluster (Corning Incorporated, Corning, NY), each well can be seeded with 1 x 106 cells of cleaved tumors or one tumor fragment in 2 ml of complete medium (CM) with IL-2 (6000 IU/ml; Chiron Corp., Emeryville, CA). In some embodiments, the tumor fragment is from about 1 mm ³ to 10 mm ³ .

In some embodiments, the first propagation culture medium is called CM, which is an abbreviation for culture medium. In some embodiments, the Stage B CM consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg/mL gentamicin. In embodiments in which cultures are initiated in 40 mL gas-permeable flasks with a 10 cm ² gas-permeable silicone bottom (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, Minn.) (Fig. 1), each flask was loaded with 10-40x106 viable cleaved tumor cells or 5-30 tumor fragments in 10-40 ml SM with IL-2 Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO ₂ and 5 days after the start of cultivation, half of the medium was removed and replaced with fresh SM and IL-2, and after 5 days, half of the medium was changed every 2-3 days.

- 40 045580

After preparation of tumor fragments, the resulting cells (ie fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, tumor digests are incubated in 2 ml wells in medium containing inactivated human AB serum (or, in some cases, as described herein, in the presence of a population of aAPC cells) with 6000 IU/ml IL-2. This population of primary cells is cultured for several days, typically 10 to 14 days, resulting in a population of total TILs, typically about 1x10 ^s total TILs. In some embodiments, the media during the first propagation contains IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, the IL-2 stock solution has a specific activity of 20-30x106 IU/mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 20x106 IU/mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 25 x 106 IU/mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a specific activity of 30 x 106 IU/mg per 1 mg vial. In some embodiments, the IL-2 stock solution has a final concentration of 4-8x106 IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5-7x106 IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6x106 IU/mg IL-2. In some embodiments, an IL-2 stock solution is prepared as described in Example 4. In some embodiments, the first propagation culture medium contains about 10,000 IU/mL IL-2, about 9,000 IU/mL IL-2, about 8,000 IU/mL IL-2, about 7000 IU/ml IL-2, about 6000 IU/ml IL-2 or about 5000 IU/ml IL-2. In some embodiments, the first culture propagation medium contains from about 9000 IU/ml IL-2 to about 5000 IU/ml IL-2. In some embodiments, the first culture propagation medium contains from about 8000 IU/ml IL-2 to about 6000 IU/ml IL-2. In some embodiments, the first culture propagation medium contains from about 7000 IU/ml IL-2 to about 6000 IU/ml IL-2. In some embodiments, the first expansion culture medium contains about 6000 IU/ml IL-2. In one embodiment, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/ml IL-2. In one embodiment, the cell culture medium further contains IL-2. In a preferred embodiment, the cell culture medium contains about 3000 IU/ml IL-2. In one embodiment, the cell culture medium contains about 1000 IU/ml, about 1500 IU/ml, about 2000 IU/ml, about 2500 IU/ml, about 3000 IU/ml, about 3500 IU/ml, about 4000 IU/ml ., about 4500 IU/ml, about 5000 IU/ml, about 5500 IU/ml, about 6000 IU/ml, about 6500 IU/ml, about 7000 IU/ml, about 7500 IU/ml or about 8000 IU/ml IL -2. In one embodiment, the cell culture medium contains from 1000 to 2000 IU/ml, from 2000 to 3000 IU/ml, from 3000 to 4000 IU/ml, from 4000 to 5000 IU/ml, from 5000 to 6000 IU/ml, from 6000 to 7000 IU/ml, 7000 to 8000 IU/ml or about 8000 IU/ml IL-2.

In some embodiments, the first culture propagation medium contains about 500 IU/ml IL-15, about 400 IU/ml IL-15, about 300 IU/ml IL-15, about 200 IU/ml IL-15, about 180 IU/ ml IL-15, about 160 IU/ml IL-15, about 140 IU/ml IL-15, about 120 IU/ml IL-15 or about 100 IU/ml IL-15. In some embodiments, the first culture propagation medium contains from about 500 IU/ml IL-15 to about 100 IU/ml IL-15. In some embodiments, the first culture propagation medium contains from about 400 IU/ml IL-15 to about 100 IU/ml IL-15. In some embodiments, the first culture propagation medium contains from about 300 IU/ml IL-15 to about 100 IU/ml IL-15. In some embodiments, the first expansion culture medium contains about 200 IU/ml IL-15. In some embodiments, the cell culture medium contains about 180 IU/ml IL-15. In one embodiment, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU/ml IL-15.

In some embodiments, the first culture propagation medium contains about 20 IU/ml IL-21, about 15 IU/ml IL-21, about 12 IU/ml IL-21, about 10 IU/ml IL-21, about 5 IU/ ml IL-21, about 4 IU/ml IL-21, about 3 IU/ml IL-21, about 2 IU/ml IL-21, about 1 IU/ml IL-21 or about 0.5 IU/ml IL- 21. In some embodiments, the first culture propagation medium contains from about 20 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the first culture propagation medium contains from about 15 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the first culture propagation medium contains from about 12 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the first culture propagation medium contains from about 10 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the first culture propagation medium contains from about 5 IU/ml IL-21 to about 1 IU/ml IL-21. In some embodiments, the first expansion culture medium contains about 2 IU/ml IL-21. In some embodiments, the cell culture medium contains about 1 IU/ml IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/ml IL-21. In one embodiment, the cell culture medium further contains IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU/ml IL-21.

In one embodiment, the cell culture medium contains the OKT-3 antibody. In some embodiments, the cell culture medium contains about 30 ng/ml OKT-3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng/ml, about 0.5 ng/ml, about 1 ng/ml, about 2.5 ng/ml, about 5 ng/ml, about 7.5 ng /ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 50 ng/ml , about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 200 ng/ml, about 500 ng/ml and about 1 μg/ml OCT antibodies -3. In one embodiment, the cell culture medium contains from 0.1 ng/ml to 1 ng/ml, from 1 ng/ml to 5 ng/ml, from 5 ng/ml to 10 ng/ml, from 10 ng/ml to 20 ng/ml, from 20 ng/ml and 30 ng/ml, from 30 ng/ml to 40 ng/ml, from 40 ng/ml to 50 ng/ml and from 50 ng/ml to 100 ng/ml OCT antibodies -3. In some embodiments, the cell culture medium does not contain the OKT-3 antibody. In some embodiments, the OKT-3 antibody is muromonab.

In some embodiments, the cell culture medium contains one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist includes a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, wherein the 4-1BB agonist is selected from the group consisting of urelumab, utomilumab, EU-101, a fusion protein, and fragments, derivatives, variants, biosimilars, and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 0.1 μg/mL to 100 μg/mL. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve a cell culture medium concentration of 20 μg/mL to 40 μg/mL.

In some embodiments, in addition to one or more TNFRSF agonists, the cell culture medium further comprises IL-2 at an initial concentration of about 3000 IU/ml and OKT-3 antibody at an initial concentration of about 30 ng/ml, wherein the one or more TNFRSF agonists includes 4-1BB agonist.

In some embodiments, the first propagation culture medium is called CM, which is an abbreviation for culture medium. In some embodiments, it is referred to as CM1 (culture medium 1). In some embodiments, the CM consists of RPMI 640 with GlutaMAX supplemented with 10% human AB serum, 25 mM HEPES, and 10 mg/mL gentamicin. In embodiments in which cultures are initiated in 40 mL gas-permeable flasks with a 10 cm ² gas-permeable silicone bottom (e.g., G-Rex10; Wilson Wolf Manufacturing, New Brighton, Minn.) (Fig. 1), each flask was loaded with 10-40x106 viable cleaved tumor cells or 5-30 tumor fragments in 10-40 ml CM with IL-2 Both G-Rex10 and 24-well plates were incubated in a humidified incubator at 37°C in 5% CO ₂ medium and after 5 days after the start of cultivation, half of the medium was removed and replaced with fresh SM and IL-2, and after 5 days, half of the medium was changed every 2-3 days. In some embodiments, the CM is CM1 as described in the examples, see Example 5. In some embodiments, the first propagation occurs in the original cell culture medium or in the first cell culture medium. In some embodiments, the initial cell culture medium or first cell culture medium contains IL-2.

In some embodiments, the first propagation process (including processes such as, for example, those described in step B of FIG. 9, which may include processes sometimes referred to as preREP) is reduced to 3-14 days, as described in the examples and figures. In some embodiments, the first propagation (including processes such as, for example, those described in step B of FIG. 9, which may include processes sometimes referred to as pre-REP) is reduced to 7-14 days, as shown in FIG. 5, 6, 8, 9, 10 and 11, and also including, for example, propagation as described in step B of FIG. 9. In some embodiments, the first propagation in stage B is reduced to 10-14 days, as shown in FIG. 5, 6, 8, 9, 10 and 11. In some embodiments, the first breeding is reduced to 11 days, as shown in FIG. 5, 6, 8, 9, 10 and 11, and also includes, for example, propagation as described in step B of FIG. 9.

In some embodiments, the first TIL propagation may continue for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first propagation of TILs may last from 1 to 14 days. In some embodiments, the first propagation of TILs may last from 2 to 14 days. In some embodiments, the first propagation of TILs may last from 3 to 14 days. In some embodiments, the first propagation of TILs may last from 4 to 14 days. In some embodiments, the first propagation of TILs may last from 5 to 14 days. In some embodiments, the first propagation of TILs may last from 6 to 14 days. In some embodiments, the first TIL expansion may last from 7 to 14

- 42 045580 days. In some embodiments, the first propagation of TILs may last from 8 to 14 days. In some embodiments, the first propagation of TILs may last from 9 to 14 days. In some embodiments, the first propagation of TILs may last from 10 to 14 days. In some embodiments, the first propagation of TILs may last from 11 to 14 days. In some embodiments, the first propagation of TILs may last from 12 to 14 days. In some embodiments, the first propagation of TILs may last from 13 to 14 days. In some embodiments, the first propagation of TILs may continue for 14 days. In some embodiments, the first propagation of TILs may last from 1 to 11 days. In some embodiments, the first propagation of TILs may last from 2 to 11 days. In some embodiments, the first propagation of TILs may last from 3 to 11 days. In some embodiments, the first propagation of TILs may last from 4 to 11 days. In some embodiments, the first propagation of TILs may last from 5 to 11 days. In some embodiments, the first propagation of TILs may last from 6 to 11 days. In some embodiments, the first propagation of TILs may last from 7 to 11 days. In some embodiments, the first propagation of TILs may last from 8 to 11 days. In some embodiments, the first propagation of TILs may last from 9 to 11 days. In some embodiments, the first propagation of TILs may last from 10 to 11 days. In some embodiments, the first TIL propagation may continue for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15 and/or IL-21 is administered as a combination during the first propagation. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21, as well as any combinations thereof, may be included during the first propagation, including, for example, during the Step B processes of FIG. 9 and as described herein. In some embodiments, the combination of IL-2, IL-15, and IL-21 is administered as a combination during the first propagation. In some embodiments, IL-2, IL-15, and IL-21, as well as any combinations thereof, may be included during the Step B processes of FIG. 9 and as described herein.

In some embodiments, the first propagation process (including processes called pre-REP; eg, step B of FIG. 9) is shortened to 3-14 days, as shown in the figures. In some embodiments, the first propagation in stage B is reduced to 7-14 days, as shown in FIG. 5, 6, 8, 9, 10 and 11. In some embodiments, the first propagation in stage B is reduced to 10-14 days, as shown in FIG. 5, 6, 8, 9, 10 and 11. In some embodiments, the first breeding is reduced to 11 days, as shown in FIG. 5, 6, 8, 9, 10 and 11.

In some embodiments, the first propagation, such as step B of FIG. 9 is performed in a closed system bioreactor. In some embodiments, a closed system is used to propagate TILs, as described herein. In some embodiments, a separate bioreactor is used. In some embodiments, the individual bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a separate bioreactor.

1. Cytokines and other additives.

The propagation methods described herein typically employ culture media containing high doses of a cytokine, particularly IL-2, as is known in the art.

Alternatively, it is further possible to use combinations of cytokines for rapid expansion and/or second expansion of TILs with combinations of two or more IL-2, IL-15 and IL-21, as described in US patent application publication No. US 2017/0107490 A1, the description of which incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, or IL-15 and IL-21, the latter being particularly useful in many embodiments implementation. The use of cytokine combinations specifically promotes the formation of lymphocytes and, in particular, T cells, as described herein.

In one embodiment, Step B may also include adding the OKT-3 antibody or muromonab to the culture medium, as described elsewhere herein. In one embodiment, Step B may also include adding a 4-1BB agonist to the culture medium, as described elsewhere herein. In one embodiment, Step B may also include adding an OX-40 agonist to the culture medium, as described elsewhere herein. In other embodiments, additives such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha agonists, including proliferator-activated receptor gamma (PPAR) agonists such as a thiazolidinedione compound, can be applied to the culture medium during Step B, as described in US Patent Application Publication No. US 2019/0307796 A1, the disclosure of which is incorporated herein by reference.

C. Stage C. Transition from the first reproduction to the second reproduction.

In some cases, the population of total TILs resulting from the first propagation, including, for example, the population of TILs obtained, for example, in step B, as shown in FIG. 9, you can

- 43 045580 immediately undergo cryopreservation using the protocols described below in this document. Alternatively, the TIL population resulting from the first propagation, referred to as the second TIL population, can be subjected to a second propagation (which may include propagations, sometimes referred to as REP), and then cryopreserved, as described below. Likewise, in the event that genetically modified TILs are to be used in therapy, a first population of TILs (sometimes referred to as a total TIL population) or a second population of TILs (which may, in some embodiments, include populations referred to as REP TIL populations) may be genetically modified to suitable treatment before propagation or after the first propagation and before the second propagation.

In some embodiments, the TILs resulting from the first propagation (eg, step B, as shown in FIG. 9) are retained until phenotyping for selection. In some embodiments, TILs obtained from the first propagation (eg, in step B, as shown in FIG. 9) are not retained and proceed directly to the second propagation. In some embodiments, the TILs obtained from the first expansion are not cryopreserved after the first expansion and before the second expansion. In some embodiments, the transition from the first propagation to the second propagation occurs about 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after how fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs about 3-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs about 4-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs about 4-10 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs about 7-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs about 14 days after fragmentation occurs.

In some embodiments, the transition from the first propagation to the second propagation occurs after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 1-14 days after fragmentation occurs. In some embodiments, the first propagation of TILs may last from 2 to 14 days. In some embodiments, the transition from the first propagation to the second propagation occurs 3-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 4-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 5-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 6-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 7-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 8-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 9-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 10-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 11-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 12-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 13-14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 14 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 1-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 2-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 3-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 4-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 5-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 6-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation is

- 44 045580 comes 7-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 8-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 9-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 10-11 days after fragmentation occurs. In some embodiments, the transition from the first propagation to the second propagation occurs 11 days after fragmentation occurs.

In some embodiments, the TILs are not retained from the first propagation until the second propagation, but the TILs proceed directly to the second propagation (eg, in some embodiments, there is no persistence from step B to step D as shown in FIG. 9). In some embodiments, the transition occurs in a closed system, as described herein. In some embodiments, TILs from the first propagation, the second population of TILs, move directly to the second propagation without a transition period.

In some embodiments, the transition from the first propagation to the second propagation, for example, step C of FIG. 9 is performed in a closed system bioreactor. In some embodiments, a closed system is used to propagate TILs, as described herein. In some embodiments, a separate bioreactor is used. In some embodiments, the individual bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a separate bioreactor.

D. Stage D. Second reproduction.

In some embodiments, the population of TIL cells is expanded after collection and initial summary processing, for example, after step A and step B, and a transition called step C, as shown in FIG. 9. This additional propagation is referred to herein as a second propagation, which may include propagation processes commonly referred to in the art as a rapid propagation process (REP, as well as those referred to in step D of FIG. 9). The second propagation is usually carried out using a culture medium containing a number of components, including feeder cells, a source of cytokines and an anti-CD3 antibody, in a gas-permeable container.

In some embodiments, the second propagation or second propagation of TILs (which may include propagations, sometimes referred to as REPs, as well as the processes identified in step D of FIG. 9) can be performed using any TIL flasks or containers known to those skilled in the art. technology. In some embodiments, the second TIL propagation may last for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL expansion may last from about 7 days to about 14 days. In some embodiments, the second expansion of TILs may last from about 8 to about 14 days. In some embodiments, the second TIL propagation may last from about 9 to about 14 days. In some embodiments, the second TIL propagation may last from about 10 days to about 14 days. In some embodiments, the second TIL propagation may last from about 11 to about 14 days. In some embodiments, the second TIL propagation may last from about 12 to about 14 days. In some embodiments, the second TIL propagation may last from about 13 to about 14 days. In some embodiments, the second expansion of the TILs may continue for about 14 days.

In one embodiment, the second propagation can be performed in a gas-permeable container by the methods of the present invention (including, for example, propagations called REP, as well as the processes specified in step D of Fig. 9). For example, TILs can be rapidly expanded by nonspecific stimulation of the T cell receptor in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15). The non-specific T cell receptor stimulus may include, for example, an anti-CD3 antibody such as about 30 ng/ml OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn , California) or UHCT-1 (commercially available from BioLegend, San Diego, California, USA). TILs can be expanded to further stimulate TILs in vitro by incorporating one or more antigens during a second expansion, including antigenic portions thereof, such as cancer epitope(s), that may optionally be expressed from a vector, such as human binding peptide leukocyte antigen A2 (HLA-A2), for example, 0.3 µM MART-1:26-35 (27 L) or gpl 00:209-217 (210 M), optionally in the presence of T-cell growth factor, such as 300 IU /ml IL-2 or IL-15. Other suitable antigens may include, for example, NY-ESO1, TRP-1, TRP-2, cancer tyrosinase antigen, MAGE-A3, SSX-2 and VEGFR2 or antigenic portions thereof. TILs can also be rapidly expanded by restimulation with the same cancer antigen(s) introduced into HLA-A2-expressing antigen-presenting cells. Alternatively, TILs can be further restimulated, for example, with irradiated autologous lymphocytes or irradiated allogeneic HLA-A2+ and IL-2 lymphocytes. In some embodiments, re-stimulation occurs as part of a second propagation. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated allogeneic HLA-A2+ and IL-2 lymphocytes.

In one embodiment, the cell culture medium further contains IL-2. In some embodiments, the cell culture medium contains about 3000 IU/ml IL-2. In one embodiment, the cell culture medium contains about 1000 IU/ml, about 1500 IU/ml, about 2000 IU/ml, about 2500 IU/ml, about 3000 IU/ml, about 3500 IU/ml, about 4000 IU/ml , about 4500 IU/ml, about 5000 IU/ml, about 5500 IU/ml, about 6000 IU/ml, about 6500 IU/ml, about 7000 IU/ml, about 7500 IU/ml or about 8000 IU/ml IL- 2. In one embodiment, the cell culture medium contains from 1000 to 2000 IU/ml, from 2000 to 3000 IU/ml, from 3000 to 4000 IU/ml, from 4000 to 5000 IU/ml, from 5000 to 6000 IU/ml, from 6000 to 7000 IU/ml, 7000 to 8000 IU/ml or about 8000 IU/ml IL-2.

In one embodiment, the cell culture medium contains an OKT3 antibody. In some embodiments, the cell culture medium contains about 30 ng/ml OKT3 antibody. In one embodiment, the cell culture medium contains about 0.1 ng/ml, about 0.5 ng/ml, about 1 ng/ml, about 2.5 ng/ml, about 5 ng/ml, about 7.5 ng /ml, about 10 ng/ml, about 15 ng/ml, about 20 ng/ml, about 25 ng/ml, about 30 ng/ml, about 35 ng/ml, about 40 ng/ml, about 50 ng/ml , about 60 ng/ml, about 70 ng/ml, about 80 ng/ml, about 90 ng/ml, about 100 ng/ml, about 200 ng/ml, about 500 ng/ml and about 1 μg/ml OKT3 antibody . In one embodiment, the cell culture medium contains from 0.1 ng/ml to 1 ng/ml, from 1 ng/ml to 5 ng/ml, from 5 ng/ml to 10 ng/ml, from 10 ng/ml to 20 ng/ml, from 20 ng/ml and 30 ng/ml, from 30 ng/ml to 40 ng/ml, from 40 ng/ml to 50 ng/ml and from 50 ng/ml to 100 ng/ml OKT3 antibodies .

In some embodiments, the combination of IL-2, IL-7, IL-15 and/or IL-21 is administered as a combination during the second propagation. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21, as well as any combinations thereof, may be included during the second propagation, including, for example, during the Step D processes of FIG. 9 and as described herein. In some embodiments, the combination of IL-2, IL-15, and IL-21 is administered as a combination during a second propagation. In some embodiments, IL-2, IL-15, and IL-21, as well as any combinations thereof, may be included during the processes of Step D of FIG. 9 and as described herein.

In some embodiments, the second expansion may be performed in a supplemented cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second propagation occurs in a cell culture medium with supplements. In some embodiments, the supplemented cell culture medium contains IL-2, OKT-3, and antigen-presenting feeder cells. In some embodiments, the second cell culture medium contains IL-2, OKT-3, and antigen presenting cells (APC; also referred to as antigen presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium containing IL-2, OKT-3, and antigen-presenting feeder cells (ie, antigen-presenting cells).

In some embodiments, the second culture propagation medium contains about 500 IU/ml IL-15, about 400 IU/ml IL-15, about 300 IU/ml IL-15, about 200 IU/ml IL-15, about 180 IU/ ml IL-15, about 160 IU/ml IL-15, about 140 IU/ml IL-15, about 120 IU/ml IL-15 or about 100 IU/ml IL-15. In some embodiments, the second culture propagation medium contains from about 500 IU/ml IL-15 to about 100 IU/ml IL-15. In some embodiments, the second culture propagation medium contains from about 400 IU/ml IL-15 to about 100 IU/ml IL-15. In some embodiments, the second culture propagation medium contains from about 300 IU/ml IL-15 to about 100 IU/ml IL-15. In some embodiments, the second culture propagation medium contains about 200 IU/ml IL-15. In some embodiments, the cell culture medium contains about 180 IU/ml IL-15. In one embodiment, the cell culture medium further contains IL-15. In a preferred embodiment, the cell culture medium contains about 180 IU/ml IL-15.

In some embodiments, the second culture propagation medium contains about 20 IU/ml IL-21, about 15 IU/ml IL-21, about 12 IU/ml IL-21, about 10 IU/ml IL-21, about 5 IU/ ml IL-21, about 4 IU/ml IL-21, about 3 IU/ml IL-21, about 2 IU/ml IL-21, about 1 IU/ml IL-21 or about 0.5 IU/ml IL- 21. In some embodiments, the second culture propagation medium contains from about 20 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the second culture propagation medium contains from about 15 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the second culture propagation medium contains from about 12 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the second culture propagation medium contains from about 10 IU/ml IL-21 to about 0.5 IU/ml IL-21. In some embodiments, the second culture propagation medium contains from about 5 IU/ml

- 46 045580

IL-21 to about 1 IU/ml IL-21. In some embodiments, the second expansion culture medium contains about 2 IU/ml IL-21. In some embodiments, the cell culture medium contains about 1 IU/ml IL-21. In some embodiments, the cell culture medium contains about 0.5 IU/ml IL-21. In one embodiment, the cell culture medium further contains IL-21. In a preferred embodiment, the cell culture medium contains about 1 IU/ml IL-21.

In some embodiments, the antigen presenting nurse cells (APCs) are PBMCs. In one embodiment, the ratio of TILs to PBMCs and/or antigen presenting cells in rapid expansion and/or second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1: 175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or about 1: 500. In one embodiment, the ratio of TILs to PBMCs during rapid expansion and/or second expansion is from 1:50 to 1:300. In one embodiment, the ratio of TILs to PBMCs during rapid expansion and/or second expansion is from 1:100 to 1:200.

In one embodiment, the REP and/or second expansion is performed in flasks with total TILs mixed with 100- or 200-fold excess of inactivated feeder cells, 30 mg/ml OKT3 anti-CD3 antibody and 3000 IU/ml IL-2 in 150 ml of medium . Media changes are made (usually 2/3 media changes by breathing fresh media) until the cells are transferred to an alternative growth chamber. Alternative growth chambers include G-REX flasks and gas permeable containers, as described in more detail below.

In some embodiments, the second propagation (which may include processes called the REP process) is reduced to 7-14 days, as described in the examples and figures. In some embodiments, the second breeding is shortened to 11 days.

In one embodiment, REP and/or second propagation can be performed using T-175 flasks and gas permeable bags as previously described (Tran, et al., J. Immunother. 2008, 31, 742-51; Dudley, et al., J. Immunother. 2003, 26, 332-42) or gas-permeable culture dishes (G-Rex flasks). In some embodiments, the second propagation (including propagations referred to as rapid propagations) is carried out in T-175 flasks, and approximately 1 x 106 TIL suspended in 150 ml of medium can be added to each T-175 flask. TILs can be cultured in a 1:1 mixture of CM and AIM-V medium supplemented with 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 antibody. T-175 flasks can be incubated at 37°C in 5% CO ₂ . Half of the media can be replaced on day 5 with 50/50 media with 3000 IU/mL IL2. In some embodiments, on day 7, cells from two T-175 flasks can be combined in a 3 liter bag and 300 ml of AIM V with 5% human AB serum and 3000 IU/ml IL-2 is added to 300 ml of TIL suspension. The number of cells in each bag was counted every day or two, and fresh medium was added to maintain the cell number between 0.5 and 2.0 x 106 cells/ml.

In one embodiment, the second propagation (which may include propagations called REPs as well as propagations specified in step D in Fig. 9) can be performed in 500 ml gas-permeable flasks with a 100 cm ² gas-permeable silicone bottom (G-Rex 100 , commercially available from Wilson Wolf Manufacturing Corporation, New Brighton, MN, USA), 5x106 or 10x106 TILs can be cultured with PBMCs in 400 ml 50/50 medium supplemented with 5% human AB serum, 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 antibody (OCT3). G-Rex 100 flasks can be incubated at 37°C in 5% CO2. On day 5, 250 ml of supernatant can be collected and placed in centrifuge tubes and centrifuged at 1500 rpm (491xg) for 10 min. The TIL pellet can be resuspended in 150 ml of fresh medium with 5% human AB serum, 3000 IU/ml IL-2 and added back to the original G-Rex 100 flasks. When TILs are serially propagated in G-Rex 100 flasks, on day 7 TILs each G-Rex 100 flask can be suspended in 300 ml of the medium present in each flask, and the cell suspension can be divided into 3 aliquots of 100 ml, which can be used to inoculate 3 G-Rex 100 flasks. 150 ml can then be added to each flask AIM-V with 5% human AB serum and 3000 IU/ml IL-2. G-Rex 100 flasks can be incubated at 37°C in 5% CO ₂ and after 4 days, 150 ml of AIM-V with 3000 IU/ml IL-2 can be added to each G-REX 100 flask. Cells can be harvested on day 14 of culture.

In one embodiment, the second expansion (including expansions called REP) is carried out in flasks with total TILs mixed with 100- or 200-fold excess of inactivated feeder cells, 30 mg/ml OKT3 anti-CD3 antibody and 3000 IU/ml IL-2 in 150 ml of medium. In some embodiments, media replacement is performed until the cells are transferred to an alternative growth chamber. In some embodiments, 2/3 of the media is replaced by breathing fresh media. In some embodiments, alternative growth chambers include G-REX flasks and gas permeable containers, as described in more detail below.

In one embodiment, a second expansion (including expansions called REP) is performed, which further includes selecting TILs for higher tumor reactivity. Any selection method known in the art can be used. For example, the methods described in US Patent Application Publication No. 2016/0010058 A1, the disclosure of which is incorporated herein by reference, can be used to select TILs for higher tumor reactivity.

Optionally, a cell viability assay can be performed after the second expansion (including expansions referred to as REP) using standard assays known in the art. For example, a trypan blue assay can be performed on a sample of total TILs, which selectively labels dead cells and allows for assessment of viability. In some embodiments, TIL samples can be counted and their viability determined using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined in accordance with the Cellometer K2 Image Cytometer automated cell counter protocol described, for example, in Example 15.

In some embodiments, second propagations (including propagations referred to as REPs) of TILs can be performed using T-175 flasks and gas permeable bags as previously described (Tran KQ, Zhou J., Durflinger KH, et al., 2008, J Immunother. , 31:742-751, and Dudley ME, Wunderlich JR, Shelton TE, et al. 2003, J Immunother., 26:332-342) or G-Rex gas permeable flasks. In some embodiments, the second propagation is carried out using flasks. In some embodiments, the second propagation is performed using G-Rex gas permeable flasks. In some embodiments, the second expansion is carried out in T-175 flasks, with about 1x106 TIL suspended in about 150 ml of medium and added to each T-175 flask. TILs were cultured with irradiated (50 Gy) allogeneic PBMCs as feeder cells in a ratio of 1:100, and the cells were cultured in a 1:1 mixture of CM and AIM-V medium (50/50 medium), supplemented with 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 antibody. T-175 flasks are incubated at 37°C in 5% CO ₂ . In some embodiments, half of the medium is replaced on day 5 with 50/50 medium with 3000 IU/ml IL-2. In some embodiments, on day 7, cells from 2 T-175 flasks are combined in a 3-liter bag and 300 ml of AIM-V with 5% human AB serum and 3000 IU/ml IL-2 is added to 300 ml of TIL suspension. The number of cells in each bag can be counted every day or two, and fresh medium can be added to maintain the cell number between about 0.5 and about 2.0 x 106 cells/ml.

In some embodiments, the second expansion (including expansions referred to as REP) is performed in 500 mL gas-permeable silicone bottom 100 cm ² flasks (GRex 100, Wilson Wolf) (Fig. 1), approximately 5x106 or 10x106 TILs are cultured with irradiated allogeneic PBMCs in a ratio of 1:100 in 400 ml of 50/50 medium supplemented with 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 antibody. G-Rex 100 flasks are incubated at 37°C in 5% CO2. In some embodiments, on day 5, 250 ml of supernatant can be collected and placed in centrifuge tubes and centrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellet can then be resuspended in 150 ml of fresh 50/50 medium with 3000 IU/ml IL-2 and added back to the original G-Rex 100 flasks. In embodiments in which TILs are serially propagated in G-Rex 100 flasks, day 7 TIL in each G-Rex 100 flask is suspended in 300 ml of the medium present in each flask and the cell suspension is divided into three 100 ml aliquots, which are used to inoculate 3 G-Rex 100 flasks. 150 ml is then added to each flask AIM-V with 5% human AB serum and 3000 IU/ml IL-2. G-Rex 100 flasks are incubated at 37°C in 5% CO ₂ and after 4 days, 150 ml of AIM-V with 3000 IU/ml IL-2 is added to each G-Rex 100 flask. Cells were harvested on day 14 of culture.

A variety of antigen receptors of T and B lymphocytes are formed as a result of somatic recombination of a limited but large number of gene segments. These gene segments include V (variability), D (diversity), J (attachment) and C (constant) determine the binding specificity and subsequent applications of immunoglobulins and T-cell receptors (TCRs). The present invention provides a method for generating TILs that exhibit and enhance T cell repertoire diversity. In some embodiments, TILs produced by the present method exhibit an increase in T cell repertoire diversity. In some embodiments, the TILs obtained from the second expansion exhibit an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is present in the immunoglobulin heavy chain. In some embodiments, the immunoglobulin variety is present in the immunoglobulin light chain. In some embodiments, diversity is present in the T cell receptor. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of alpha, beta, gamma and delta receptors. In some embodiments, an increase in T cell receptor (TCR) alpha and/or beta expression is observed. In some embodiments, an increase in T cell receptor (TCR) alpha expression is observed. In some embodiments, an increase in T cell receptor (TCR) beta expression is observed. In some embodiments, an increase in TCRab (ie, TCRa/β) expression is observed.

In some embodiments, a second propagation culture medium (e.g.

- 48 045580 sometimes called CM2 or second cell culture medium) contains IL-2, OKT-3, and antigen presenting feeder cells (APC), as described in more detail below.

In some embodiments, a second propagation, such as step D of FIG. 9 is performed in a closed system bioreactor. In some embodiments, a closed system is used to propagate TILs, as described herein. In some embodiments, a separate bioreactor is used. In some embodiments, the individual bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a separate bioreactor.

1. Nutrient cells and antigen presenting cells.

In one embodiment, the second expansion procedures described herein (e.g., including expansions such as those described in step D of FIG. 9, as well as procedures referred to as REP) require an excess of nutritional cells during REP TIL expansion and/or during time of second reproduction. In many embodiments, the nutritional cells are peripheral blood mononuclear cells (PBMCs) derived from standard units of whole blood from healthy blood donors. PBMC are obtained using standard methods such as Ficoll-Paque gradient separation.

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in REP procedures as described in the Examples, particularly Example 14, which provides an exemplary protocol for assessing the replication failure of irradiated allogeneic PBMCs.

In some embodiments, PBMCs are considered replication-incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells on day 14 is less than the initial number of viable cells placed in culture on day 0 REP and/or day 0 second breeding (i.e. the day the second breeding begins), see, for example, example 14.

In some embodiments, PBMCs are considered replication-incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells cultured in the presence of OKT3 and IL-2 at day 7 and day 14 has not increased from baseline the number of viable cells placed in culture on day 0 REP and/or day 0 of the second propagation (i.e., the day the second propagation begins). In some embodiments, PBMCs are cultured in the presence of 30 ng/ml OKT3 antibody and 3000 IU/ml IL-2, see, for example, Example 13.

In some embodiments, PBMCs are considered replication-incompetent and accepted for use in the TIL expansion procedures described herein if the total number of viable cells cultured in the presence of OKT3 and IL-2 at day 7 and day 14 has not increased from baseline the number of viable cells placed in culture on day 0 REP and/or day 0 of the second propagation (i.e., the day the second propagation begins). In some embodiments, PBMCs are cultured in the presence of 5-60 ng/ml OKT3 antibody and 1000-6000 IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 10-50 ng/ml OKT3 antibody and 2000-5000 IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 20-40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In some embodiments, PBMCs are cultured in the presence of 25-35 ng/ml OKT3 antibody and 2500-3500 IU/ml IL-2.

In some embodiments, the antigen-presenting nurse cells are PBMCs. In some embodiments, the antigen-presenting nutritional cells are artificial antigen-presenting nutritional cells. In one embodiment, the ratio of TILs to antigen presenting nurse cells in the second expansion is about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400 or about 1:500. In one embodiment, the ratio of TILs to antigen-presenting nurse cells in the second expansion is from 1:50 to 1:300. In one embodiment, the ratio of TILs to antigen-presenting nurse cells in the second expansion is from 1:100 to 1:200.

In one embodiment, the second expansion procedures described herein require a ratio of about 2.5 x 109 nutrient cells to about 100 x 106 TILs. In another embodiment, the second expansion procedures described herein require a ratio of about 2.5 x 109 feeder cells to about 50 x 106 TILs. In yet another embodiment, the second expansion procedures described herein require a ratio of about 2.5x109 nutrient cells to about ^25x106 TIL.

In one embodiment, the second propagation procedures described herein require an excess of nutritional cells during the second propagation. In many embodiments, the nutritional cells are peripheral blood mononuclear cells (PBMCs) derived from standard units of whole blood from healthy blood donors. PBMCs are obtained from

- 49 045580 using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting cells (aAPC) are used instead of PBMCs.

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the TIL propagation procedures described herein, including the exemplary procedures described in FIG. 5, 6, 8, 9, 10 and 11.

In one embodiment, artificial antigen presenting cells are used in a second expansion as a replacement for or in combination with PBMCs.

2. Cytokines.

The propagation methods described herein typically employ culture media containing high doses of a cytokine, particularly IL-2, as is known in the art.

Alternatively, it is further possible to use cytokine combinations for rapid expansion and/or second expansion of TILs with combinations of two or more IL-2, IL-15 and IL-21, as generally described in international publication no. WO 2015/189356 and international publication W no. WO 2015/189357, which are expressly incorporated herein by reference in their entirety. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, the latter being particularly useful in many embodiments implementation. The use of cytokine combinations specifically promotes the formation of lymphocytes and, in particular, T cells, as described herein.

3. Antibodies to CD3.

In some embodiments, the culture medium used in the propagation methods described herein (including those referred to as REP, see, for example, FIG. 9) also includes an anti-CD3 antibody. Anti-CD3 antibody in combination with IL-2 causes T cell activation and cell division in the TIL population. This effect can be seen with full-length antibodies, as well as Fab and F(ab') ₂ fragments, with the former usually being preferred; see, for example, Tsoukas et al., J. Immunol. 1985, 135, 1719, which is incorporated herein by reference in its entirety.

As will be appreciated by those skilled in the art, there are a number of suitable anti-human CD3 antibodies that find use in the invention, including polyclonal and monoclonal anti-human CD3 antibodies from various mammals, including, but not limited to, mouse, human, primate, rat and dogs. In particular embodiments, the anti-CD3 antibody OKT3 (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, CA) is used.

E. Step F: TIL Collection.

After the second stage of propagation, the cells can be collected. In some embodiments, TILs are collected after one, two, three, four or more propagation steps, for example, as shown in FIG. 9. In some embodiments, TILs are collected after two propagation steps, for example, as shown in FIG. 9.

TIL can be collected by any suitable and sterile method, including, for example, centrifugation. Methods for collecting TILs are well known in the art, and any such known methods can be used in the present process. In some embodiments, TILs are collected using an automated system.

Cell harvesters and/or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, Inc. Any cell harvester can be used with the present methods. In some embodiments, the cell harvester and/or cell processing systems are membrane-based cell harvesters. In some embodiments, cell collection is accomplished using a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term LOVO cell processing system also refers to any instrument or device, manufactured by any supplier, that can pump a solution containing cells through a membrane or filter, such as a rotating membrane or rotating filter, in a sterile and/or closed system environment, allowing Processing cells in continuous flow mode removes the supernatant or cell culture medium without forming a precipitate. In some embodiments, the cell harvester and/or cell processing system may perform the separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, collection, such as step E of FIG. 9 is performed from a closed system bioreactor. In some embodiments, a closed system is used to propagate TILs, as described herein. In some embodiments, a separate bioreactor is used. In some embodiments, the individual bioreactor used is, for example, G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a separate bioreactor.

F. Step F. Final formulation/transfer to infusion bag.

After completing steps A-E, illustrated in illustrative order in FIG. 9 and in detail

- 50 045580 described above and herein, the cells are transferred into a container for use when administered to a patient. In some embodiments, once a therapeutically sufficient amount of TIL has been obtained by the propagation methods described above, they are transferred to a container for use when administered to a patient.

In one embodiment, TILs propagated using the APCs of the present invention are administered to a patient in the form of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present invention can be administered by any suitable method known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts from about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic.

1. Pharmaceutical compositions, dosages and dosage regimens.

In one embodiment, TILs propagated by the methods of the present invention are administered to a patient in the form of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TILs in a sterile buffer. TILs expanded using PBMCs of the present invention can be administered by any suitable method known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts from about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable dose of TIL may be administered. In some embodiments, from about 2.3 x 10 ¹⁰ to about 13.7 x 10 10 TIL are administered, with an average of about 7.8 x 10 10 TIL, especially if the cancer is melanoma. In one embodiment, from about 1.2x1010 to about ^4.3x1010 TIL is administered. In some embodiments, from about 3 x 10 ¹⁰ to about 12 x 10 ¹⁰ TIL are administered. In some embodiments, from about 4x1010 to about 10x10 ¹⁰ TIL are administered. In some embodiments, from about 5x1010 to about 8x10 ¹⁰ TIL are administered. In some embodiments, from about 6x1010 to about 8x10 ¹⁰ TIL are administered. In some embodiments, from about 7x10 ¹⁰ to about 8x10 ¹⁰ TIL are administered. In some embodiments, the therapeutically effective dose is from about 2.3 x 10 ¹⁰ to about 13.7 x 10 10 . In some embodiments, the therapeutically effective dose is about 7.8 x 10 ¹⁰ TIL, especially if the cancer is melanoma. In some embodiments, the therapeutically effective dose is from about 1.2x10 ¹⁰ to about 4.3x10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is from about 3x10 ¹⁰ to about 12x10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is from about 4x10 ¹⁰ to about 10x10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is from about 5x10 ¹⁰ to about 8x10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is from about 6x10 ¹⁰ to about 8x10 ¹⁰ TIL. In some embodiments, the therapeutically effective dose is from about 7x10 ¹⁰ to about 8x10 ¹⁰ TIL.

In some embodiments ^, the number of TILs provided in the pharmaceutical compositions of the present invention is about 1x106, 2x106, 3x106, 4x106, 5x106, 6x106, 7x106, 8x106, 9x106, 1x107, ^2x107 , ^3x107 , ^4x107 , 5x10 ⁷ , 6x10 ⁷ , 7x10 ⁷ , 8x10 ⁷ , 9x10 ⁷ , 1x10 ⁸ , 2x10 8, 3x10 ⁸ , 4x10 ⁸ , 5x10 ⁸ , 6x10 ⁸ , 7x10 ⁸ , 8x108, ^{9x10 8 ,} 1x109, 2 x109, 3x109, 4x109, 5x109, 6x109, 7x109, 8x109, 9x109, 1x10 ^{10 ,} 2x10 10, 3x10 ¹⁰ , 4x10 10, 5x10 ¹⁰ , 6x10 ¹⁰ , 7x10 ¹⁰ , 8x10 ¹⁰ , 9x10 ¹⁰ , 1x1011, 2x10 ¹¹ , 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 8x1011, 9x1011 , 1x10 ¹² , 2x10 ¹² , 3x10 ¹² , 4x10 12 , 5x10 ¹² , 6x10 ¹² , 7x10 ¹² , 8x10 ¹² , 9x10 ¹² , 1x10 ¹³ , ^{2x10 13} , 3x10 ¹³ , 4 x10 ¹³ , 5x10 13 , 6x10 ¹³ , ^{7x10 13 ,} 8x10 ¹³ and 9x10 ¹³ . In one embodiment, the number of TILs provided in the pharmaceutical compositions of the present invention ranges from 1x106 to 5x106, 5x106 to ^1x107 , ^1x107 to 5x107, ^5x107 to ^1x108 , 1x108 to 5x108, 5x108 to 1x109, from 1x109 to 5x109, from 5x109 to 1x10 ¹⁰ , from 1x10 ¹⁰ to 5x10 ¹⁰ , from 5x10 ¹⁰ to 1x1011, from 5x1011 to 1x10 ¹² , from 1x10 ¹² to 5x10 ¹² and from 5x10 ¹ 2 to 1x10 ¹³ .

In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% , 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0 .05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001% , 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w. , wt./vol. or v/v pharmaceutical composition.

In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50% , 19.25%, 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16, 50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13 .50%, 13.25%

- 51 045580

13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25 %, 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7 ,25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1 ,50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003% , 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001 % w/w, w/v or v/v pharmaceutical composition.

In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention ranges from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, or about 1% to about 10% w/w, w/v or v/v pharmaceutical composition.

In some embodiments, the concentration of TILs provided in the pharmaceutical compositions of the present invention ranges from about 0.001% to about 10%, from about 0.01% to about 5%, from about 0.02% to about 4.5% , about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07 % to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v . or v/v pharmaceutical composition.

In some embodiments, the amount of TIL provided in the pharmaceutical compositions of the present invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g , 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g , 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g , 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g , 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0, 0004 g, 0.0003 g, 0.0002 g or 0.0001 g.

In some embodiments, the amount of TIL provided in the pharmaceutical compositions of the present invention is greater than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0, 09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g , 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1 .5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8g, 8.5g, 9g, 9.5g or 10g.

The TILs provided in the pharmaceutical compositions of the present invention are effective over a wide range of dosages. The exact dosage will depend on the route of administration, the form in which the compound is administered, the sex and age of the subject being treated, the body weight of the subject, and the preference and experience of the attending physician. If necessary, clinically established doses of TIL can also be used. The amounts of pharmaceutical compositions administered using the methods described herein, such as TIL doses, will depend on the human or mammal being treated, the severity of the disease or condition, the rate of administration, the disposition of the active pharmaceutical ingredients, and the discretion of the attending physician.

In some embodiments, the TIL may be administered as a single dose. Such administration may be by injection, for example intravenous injection. In some embodiments, TILs can be administered in multiple doses. Dosing can be done once, twice, three times, four times, five times, six times or more than six times a year. Dosing can be done once a month, once every two weeks, once a week or once every other day. The administration of TIL can continue for as long as necessary.

In some embodiments, the effective dose of TIL is about ^1x106 , ^2x106 , 3x106, ^4x106 , ^5x106 , ^6x106 , ^7x106 , ^8x106 , ^9x106 , ^1x107 , ^2x107 , ^3x107 , 4x ^{10 7 ,} 5x10 ⁷ , 6x10 ⁷ , 7x10 ⁷ , 8x10 ⁷ , 9x10 ⁷ ,

1x10 ^{8 ,} 2x10 8 ^, 3x10 ⁸ , 4x10 ⁸ , 5x10 ⁸ , 6x10 ⁸ , 7x10 ⁸ , 8x10 ⁸ , 9x10 ⁸ , 1x10 ⁹ , 2x10 9, 3x10 ⁹ , 4x10 ⁹ , 5x10 ⁹ , 6x10 ⁹ ,

7x10 ⁹ , 8x10 ⁹ , 9x10 ⁹ , 1x10 ¹⁰ , 2x10 10, 3x10 ¹⁰ , 4x10 ^{10, 5x10 10 , 6x10 10, 7x10 10, 8x10 10 , 9x10 10 ,} 1x1011 , 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011 ^, 8x1011, 9x1011, 1x10 ¹² , 2x10 12, 3x10 ¹² , 4x10 12, 5x10 ¹² , 6x10 ¹² , 7x10 ¹² , 8x10 ¹² , 9x10 ¹² , 1x10 ¹³ , 2x10 13 , 3x10 ¹³ , 4x10 ^{13 ,} 5x10 ^{13 ,} 6x10 ^{13 ,} 7x10 ¹³ , 8x10 ¹³ , and 9x10 ¹³ . In some embodiments, the effective dose of TIL is in the range of 1x10 ⁶ to 5x10 ⁶ , 5x10 ⁶ to 1x10 ⁷ , 1x10 ⁷ to 5x10 ⁷ , 5x10 ⁷ to 1x10 ⁸ , 1x10 ⁸ to 5x10 ⁸ , 5x10 ⁸ to 1x10 ⁹ , from 1x10 ⁹ to 5x10 ⁹ , from 5x10 ⁹ to 1x10 ¹⁰ , from 1x10 ¹⁰ to 5x10 ¹⁰ , from 5x10 ¹⁰ to 1x1011, from 5x1011 to 1x10 ¹² , from 1x10 ¹² to 5x10 ¹² and from 5x10 ¹² to 1x10 ¹³ .

In some embodiments, the effective dose of TIL is in the range from about 0.01 mg/kg to about 4.3 mg/kg, from about 0.15 mg/kg to about 3.6 mg/kg, from about 0.3 mg /kg to about 3.2 mg/kg, from

- 52 045580 about 0.35 mg/kg to about 2.85 mg/kg, from about 0.15 mg/kg to about 2.85 mg/kg, from about 0.3 mg/kg to about 2.15 mg /kg, from about 0.45 mg/kg to about 1.7 mg/kg, from about 0.15 mg/kg to about 1.3 mg/kg, from about 0.3 mg/kg to about 1.15 mg/kg, from about 0.45 mg/kg to about 1 mg/kg, from about 0.55 mg/kg to about 0.85 mg/kg, from about 0.65 mg/kg to from about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg /kg, from about 1 mg/kg to about 1.85 mg/kg, from about 1.15 mg/kg to about 1.7 mg/kg, from about 1.3 mg/kg to about 1.6 mg/ kg, from about 1.35 mg/kg to about 1.5 mg/kg, from about 2.15 mg/kg to about 3.6 mg/kg, from about 2.3 mg/kg to about 3.4 mg /kg, from about 2.4 mg/kg to about 3.3 mg/kg, from about 2.6 mg/kg to about 3.15 mg/kg, from about 2.7 mg/kg to about 3 mg/ kg, from about 2.8 mg/kg to about 3 mg/kg, or from about 2.85 mg/kg to about 2.95 mg/kg.

In some embodiments, the effective dose of TIL ranges from about 1 mg to about 500 mg, from about 10 mg to about 300 mg, from about 20 mg to about 250 mg, from about 25 mg to about 200 mg, from about 1 mg to about 50 mg, from about 5 mg to about 45 mg, from about 10 mg to about 40 mg, from about 15 mg to about 35 mg, from about 20 mg to about 30 mg, from about 23 mg to about 28 mg, from about 50 mg to about 150 mg, from about 60 mg to about 140 mg, from about 70 mg to about 130 mg, from about 80 mg to about 120 mg, from about 90 mg to about 110 mg, or from about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, from about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of TIL can be administered as a single or multiple dose by any of the conventional routes of administration of agents having similar properties, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, transplantation or inhalation.

G. Optional Cell Viability Assays.

Optionally, a cell viability assay can be performed after the first propagation in step B using standard assays known in the art. For example, a trypan blue assay can be performed on a sample of total TILs, which selectively labels dead cells and allows for assessment of viability. Other assays for viability testing may include, but are not limited to, the Alamar Blue assay and the MTT assay.

1. Cell count, viability, flow cytometry.

In some embodiments, cell number and/or viability are measured. The expression of markers, such as, but not limited to, CD3, CD4, CD8 and CD56, as well as any others disclosed or described herein, can be measured by flow cytometry with antibodies, such as, but not limited to, those commercially available from from BD Biosciences (BD Biosciences, San Jose, CA) using a FACSCanto™ flow pitometer (BD Biosciences). Cells can be counted manually using a disposable C-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed by any method known in the art, including, but not limited to, trypan blue staining.

In some cases, the total TIL population can be cryopreserved immediately using the protocols described below. Alternatively, the total TIL population can be REPed and then cryopreserved as described below. Likewise, in the event that genetically modified TILs are to be used in a therapy, populations of total or REP TILs can be genetically modified for appropriate treatment.

2. Cell cultures.

In one embodiment, a method of propagating TILs may include using about 5,000 ml to about 25,000 ml of cell culture medium, from about 5,000 ml to about 10,000 ml of cell culture medium, or from about 5,800 ml to about 8,700 ml of cell culture medium. In one embodiment, no more than one type of cell culture medium is used to propagate TILs. Any suitable cell culture medium can be used, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, Calif.). In this regard, the patented methods have the advantage of reducing the amount of media, as well as the number of types of media required for TIL propagation. In one embodiment, TIL propagation may involve adding fresh cell culture medium to the cells (also called cell feeding) no more frequently than every third or fourth day. Propagating cells in a gas-permeable container simplifies the procedures required for cell propagation by reducing the frequency of feeding required for cell propagation.

In an embodiment, the cell culture medium in the first and/or second gas-permeable container is unfiltered. The use of unfiltered cell media can simplify the procedures required for cell propagation. In one embodiment, beta-mercaptoethanol (BME) is absent from the cell environment in the first and/or second gas-permeable container.

In one embodiment, the duration of the method includes obtaining a sample of tumor tissue from a mammal; culturing a tumor tissue sample in the first gas-permeable

- 53 045580 container containing cell medium; obtaining TIL from a tumor tissue sample; reproduction

TIL in a second gas-permeable container containing cell media using aAPC for a period of about 14 to about 42 days, such as about 28 days.

In one embodiment, TILs are propagated in gas-permeable containers. Gas-permeable containers have been used to propagate TILs using PBMCs using methods, compositions and devices known in the art, including those described in US Patent Application Publication No. 2005/0106717 A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are propagated in gas-permeable bags. In one embodiment, TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are propagated using a cell expansion system that propagates TILs in gas-permeable bags, such as the WAVE bioreactor system, also known as the Xuri W5 cell expansion system (GE Healthcare). In one embodiment, the cell propagation system includes a gas-permeable cell bag with a volume selected from the group consisting of about 100 ml, about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml, about 700 ml, about 800 ml, about 900 ml, about 1 l, about 2 l, about 3 l, about 4 l, about 5 l, about 6 l, about 7 l, about 8 l, about 9 l and about 10 l. In one embodiment, TILs can be propagated in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such embodiments allow the cell population to be expanded from about 5x105 cells/ ^cm2 to 10x106 to 30x106 cells/ ^cm2 . In one embodiment, said propagation is carried out without adding fresh cell culture medium to the cells (also called cell feeding). In one embodiment, this occurs without feeding while the medium is at a height of about 10 cm in the G-Rex flask. In one embodiment, this occurs without feeding, but with the addition of one or more cytokines. In one embodiment, the cytokine can be added as a bolus without the need to mix the cytokine with the medium. Such containers, devices and methods are known in the art and have been used for propagating TILs, including those described in US Patent Application Publication No. US 2014/0377739A1, International Publication No. WO 2014/210036 A1, US Patent Application Publication No. US 2013/0115617 A1, International Publication No. WO 2013/188427 A1, US Patent Application Publication No. US 2011/0136228 A1, US Patent No. US 8809050 B2, International Publication No. WO 2011/072088 A2, US Patent Application Publication No. US 2016/0208216 A1 , publication of US patent application No. US 2012/0244133 A1, international publication No. WO 2012/129201 A1, publication of US patent application No. US 2013/0102075 A1, publication of US patent application No. US 8956860 B2, international publication No. WO 2013/ 173835 A1, US Patent Application Publication No. US 2015/0175966 A1, the disclosure of which is incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292. Optional genetic modification of TILs

In some embodiments, TILs are optionally genetically modified to include additional functional properties, including, but not limited to, a high-affinity T cell receptor (TCR), e.g., a TCR targeting a tumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen receptor (CAR) that binds to a tumor-associated cell surface molecule (eg, mesothelin) or a lineage-specific cell surface molecule (eg, CD 19).

H. Optional cryopreservation of TILs.

As described above and exemplified in steps A-E in FIG. 9, cryopreservation can be performed at various points in time throughout the TIL propagation process. In some embodiments, the expanded population of TILs may be cryopreserved after a second expansion (as shown, for example, for step D in FIG. 9). Cryopreservation can generally be accomplished by placing the TIL population in a freezing solution, for example, containing 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). Cells in solution are placed in cryogenic vials and stored for 24 hours at -80°C with optional transfer to nitrogen gas freezers for cryopreservation, see Sadeghi, et al., Acta Oncologica 2013, 52, 978-986. In some embodiments, TILs are cryopreserved in 5% DMSO. In some embodiments, the TILs are cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, TILs are cryopreserved by the methods described in Examples 8 and 9.

If necessary, the cells are removed from the freezer and thawed at 37°C in a water bath until approximately 4/5 of the solution is thawed. Cells are typically resuspended in complete medium and optionally washed one or more times. In some embodiments, thawed TILs can be counted and assessed for viability, as is known in the art.

I. Phenotypic characteristics of expanded TILs.

In some embodiments, TILs are analyzed for expression of a variety of phenotypic markers after expansion, including those described herein and in the Examples. In one version

- 54 045580 implementations examine the expression of one or more phenotypic markers. In some embodiments, the phenotypic characteristics of the TILs are analyzed after the first propagation in step B. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transition in step C. In some embodiments, the phenotypic characteristics of the TILs are analyzed during the transition in accordance with step C and after cryopreservation. In some embodiments, the phenotypic characteristics of the TILs are analyzed after a second propagation in accordance with step D. In some embodiments, the phenotypic characteristics of the TILs are analyzed after two or more propagations in accordance with step D. In some embodiments, the marker is selected from the group consisting of TCRab (t i.e. TCRa/β), CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38 and HLA-DR. In some embodiments, the marker is selected from the group consisting of TCRab (ie, TCRa/β), CD57, CD28, CD4, CD27, CD56, and CD8a. In one embodiment, the marker is selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38 and HLA-DR. In some embodiments, the expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen markers is examined. In some embodiments, the expression of one or more markers from each group is examined. In some embodiments, the expression of one or more of HLA-DR, CD38, and CD69 is preserved (ie, does not show a statistically significant difference) in fresh TILs compared to thawed TILs. In some embodiments, the activation status of the TIL is maintained in the thawed TIL.

In one embodiment, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD-1, and TIM-3. In some embodiments, the regulatory marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, expression of regulatory molecules is reduced in thawed TILs compared to fresh TILs. In some embodiments, expression of the regulatory molecules LAG-3 and TIM-3 is reduced in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in the expression of CD4, CD8, NK, TCRae. In some embodiments, there is no significant difference in the expression of CD4, CD8, NK, TCRae and/or memory markers in fresh TILs compared to thawed TILs. In some embodiments, there is no significant difference in the expression of CD4, CD8, NK, TCRae between TILs obtained through the methods presented herein, as shown, for example, in FIG. 9, and/or TILs produced by methods other than those presented herein, including, for example, methods other than those presented in FIG. 9.

In some embodiments, selection of a first TIL population, a second TIL population, a third TIL population, a harvested TIL population, and/or a therapeutic TIL population based on CD4, CD8, and/or NK expression, TCRae is not performed during any of the steps, including those described above or shown, for example, in Fig. 9. In some embodiments, selection of the first population of TILs based on CD4, CD8 and/or NK TCRae expression is not performed. In some embodiments, selection of the second population of TILs based on expression of CD4, CD8 and/or NK, TCRae is not performed. In some embodiments, selection of the third TIL population based on CD4, CD8 and/or NK, TCRae expression is not performed. In some embodiments, selection of the collected TIL population based on CD4, CD8 and/or NK, TCRae expression is not performed. In some embodiments, selection of a TIL therapeutic population based on CD4, CD8 and/or NK, TCRae expression is not performed.

In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, or a harvested TIL population based on the expression of CD4, CD8 and/or NK, TCRae is not performed in any of the steps (a)-(f) of the tumor infiltrating method lymphocytes (TIL) into the therapeutic TIL population, including:

(a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to a closed system;

(c) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system;

(d) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to obtain

- 55 045580 third TIL population, wherein the third TIL population is a therapeutic population

TIL, which includes an increased subpopulation of effector T cells and/or central memory T cells compared to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step ( d) occurs without opening the system;

(e) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system.

In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, or a pooled TIL population based on the expression of CD4, CD8 and/or NK, TCRof is not performed in any of steps (a)-(h) of the method of treating a subject. a cancer patient, the method comprising administering expanded tumor infiltrating lymphocytes (TILs) and comprising:

(a) obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) adding tumor fragments to a closed system;

(c) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system;

(d) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third a population of TILs, wherein the third population of TILs is a therapeutic population of TILs that includes an increased subpopulation of effector T cells and/or central memory T cells compared to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system;

(e) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening;

(g) optionally cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process; and (h) administering to the patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In some embodiments, the memory marker is selected from the group consisting of CCR7 and CD62L.

In some embodiments, the viability of fresh TILs compared to thawed TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, the viability of both fresh and thawed TILs is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98%. In some embodiments, both fresh and thawed product viability is greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89% or more than 90%. In some embodiments, both fresh and thawed product viability is greater than 86%.

In one embodiment, re-stimulated TILs can also be assessed for cytokine release using cytokine release assays. In some embodiments, TILs can be assessed for interferon-7 (IFN-7) secretion in response to either OKT3 stimulation or co-culture with autologous tumor digestion. For example, in embodiments that employ OKT3 stimulation, the TILs are thoroughly washed and duplicate wells are prepared with 1x105 cells in 0.2 mL SM in 96-well flat-bottom plates precoated with 0.1 or 1.0 μg/mL OKT3 , diluted in phosphate-buffered saline. After overnight incubation, supernatants were collected and IFN-7 in the supernatant was measured by ELISA (Pierce/Endogen, Woburn, MA). For the co-culture assay, 1x105 TIL cells are plated in a 96-well plate with autologous tumor cells. (Ratio 1:1). After a 24-hour incubation, the supernatants are collected and IFN-7 release can be quantified, for example, using ELISA.

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Flow Cytometric Analysis of Cell Surface Biomarkers: TIL samples were aliquoted for flow cytometric analysis of cell surface markers, see Examples 7, and 9.

In some embodiments, TILs are assessed for the presence of various regulatory markers. In some embodiments, the regulatory marker is selected from the group consisting of TCR α/β, CD56, CD27, CD28, CD57, CD45RA, CD45RO, CD25, CD127, CD95, IL-2R-, CCR7, CD62L, KLRG1, and CD122. In some embodiments, the regulatory marker is a TCR α/β. In some embodiments, the regulatory marker is CD56. In some embodiments, the regulatory marker is CD27. In some embodiments, the regulatory marker is CD28. In some embodiments, the regulatory marker is CD57. In some embodiments, the regulatory marker is CD45RA. In some embodiments, the regulatory marker is CD45RO. In some embodiments, the regulatory marker is CD25. In some embodiments, the regulatory marker is CD127. In some embodiments, the regulatory marker is CD95. In some embodiments, the regulatory marker is IL-2R-. In some embodiments, the regulatory marker is CCR7. In some embodiments, the regulatory marker is CD62L. In some embodiments, the regulatory marker is KLRG1. In some embodiments, the regulatory marker is CD122.

In one embodiment, the expanded TILs are analyzed for the expression of a variety of phenotypic markers, including those described herein and in the Examples. In one embodiment, the expression of one or more phenotypic markers is examined. In some embodiments, the marker is selected from the group consisting of TCRab (ie, TCRa/β), CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, and HLA-DR. In some embodiments, the marker is selected from the group consisting of TCRab (ie, TCRa/β), CD57, CD28, CD4, CD27, CD56, and CD8a. In one embodiment, the marker is selected from the group consisting of CD45RA, CD8a, CCR7, CD4, CD3, CD38 and HLA-DR. In some embodiments, the expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen markers is examined. In some embodiments, the expression of one or more markers from each group is examined. In some embodiments, the expression of one or more of HLA-DR, CD38, and CD69 is preserved (ie, does not show a statistically significant difference) in fresh TILs compared to thawed TILs. In some embodiments, the activation status of the TILs is maintained in the thawed TILs.

In one embodiment, the expression of one or more regulatory markers is measured. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, the regulatory marker is selected from the group consisting of CD137, CD8a, Lag3, CD4, CD3, PD1, and TIM-3. In some embodiments, the regulatory marker is selected from the group consisting of CD69, CD8a, TIGIT, CD4, CD3, KLRG1, and CD154. In some embodiments, expression of regulatory molecules is reduced in thawed TILs compared to fresh TILs. In some embodiments, expression of the regulatory molecules LAG-3 and TIM-3 is reduced in thawed TILs compared to fresh TILs. In some embodiments, there is no significant difference in the expression of CD4, CD8, NK, TCRαβ. In some embodiments, there is no significant difference in the expression of CD4, CD8, NK, TCRaβ and/or memory markers in fresh TILs compared to thawed TILs.

In some embodiments, the memory marker is selected from the group consisting of CCR7 and CD62L.

In some embodiments, the viability of fresh TILs compared to thawed TILs is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. In some embodiments, the viability of both fresh and thawed TILs is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 98%. In some embodiments, both fresh and thawed product viability is greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89% or more than 90%. In some embodiments, both fresh and thawed product viability is greater than 86%.

In one embodiment, re-stimulated TILs can also be assessed for cytokine release using cytokine release assays. In some embodiments, TILs can be assessed for interferon-7 (IFN-7) secretion in response to either OKT3 stimulation or co-culture with autologous tumor digestion. For example, in embodiments that employ OKT3 stimulation, the TILs are thoroughly washed and duplicate wells are prepared with 1 x 10 ⁵ cells in 0.2 ml of SM in 96-well flat-bottom plates pre-coated with 0.1 or 1. 0 µg/ml OKT3 diluted in phosphate-buffered saline. After overnight incubation, supernatants were collected and IFN-7 in the supernatant was measured by ELISA (Pierce/Endogen, Woburn, MA). For the co-culture assay, 1 x 10 ⁵ TIL cells are plated in a 96-well plate with autologous tumor cells. (Ratio 1:1). After a 24-hour incubation, the supernatants are collected and IFN-7 release can be quantified, for example, using ELISA.

In some embodiments, TILs that exhibit granzyme B secretion greater than 3,000 pg/10 ⁶ TIL to 300,000 pg/10 ⁶ TIL are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 3000 pg/10 ⁶ TIL, greater than 5000 pg/10 ⁶ TIL, greater than 7000 pg/10 ⁶ TIL , more than 9000 pg/10 ⁶ TIL, more than 11000 pg/10 ⁶ TIL, more than 13000 pg/10 ⁶ TIL, more than 15000 pg/10 ⁶ TIL, more than 17000 pg/10 ⁶ TIL, more than 19000 pg/10 ⁶ TIL, more 20,000 pg/10 ⁶ TIL, more than 40,000 pg/10 ⁶ TIL, more than 60,000 pg/10 ⁶ TIL, more than 80,000 pg/10 ⁶ TIL, more than 100,000 pg/10 ⁶ TIL, more than 120,000 pg/10 ⁶ TIL, more than 140,000 pg /10 ⁶ TIL, more than 160,000 pg/10 ⁶ TIL, more than 180,000 pg/10 ⁶ TIL, more than 200,000 pg/10 ⁶ TIL, more than 220,000 pg/10 ⁶ TIL, more than 240,000 pg/10 ⁶ TIL, more than 260,000 pg/10 ⁶ TILs greater than 280,000 pg/10 ⁶ TILs greater than 300,000 pg/10 ⁶ TILs or greater are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 3000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 5000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 7000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 9000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 11,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 13,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 15,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 17,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 19,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 20,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 40,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 60,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 80,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 100,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 120,000 pg/10 ⁶ TIL, representing

- 58 045580 are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 140,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 160,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 180,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 200,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 220,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 240,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 260,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 280,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 300,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion from greater than 3,000 pg/10 ⁶ TIL to 300,000 pg/10 ⁶ TIL are TILs produced by propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 3000 pg/10 ⁶ TIL, greater than 5000 pg/10 ⁶ TIL, greater than 7000 pg/10 ⁶ TIL , more than 9000 pg/10 ⁶ TIL, more than 11000 pg/10 ⁶ TIL, more than 13000 pg/10 ⁶ TIL, more than 15000 pg/10 ⁶ TIL, more than 17000 pg/10 ⁶ TIL, more than 19000 pg/10 ⁶ TIL, more 20000 pg/10 ⁶ TIL, more than 40000 pg/10 ⁶ TIL, more

60,000 pg/10 ⁶ TIL, more than 80,000 pg/10 ⁶ TIL, more than 100,000 pg/10 ⁶ TIL, more than 120,000 pg/10 ⁶ TIL, more

140,000 pg/10 ⁶ TIL, more than 160,000 pg/10 ⁶ TIL, more than 180,000 pg/10 ⁶ TIL, more than 200,000 pg/10 ⁶ TIL, more

220,000 pg/10 ⁶ TIL, more than 240,000 pg/10 ⁶ TIL, more than 260,000 pg/10 ⁶ TIL, more than 280,000 pg/10 ⁶ TIL, more

300,000 pg/10 ⁶ TIL or more are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 3000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 5000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 7000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 9000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 11,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 13,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 15,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 17,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B, and/or 2C, and/or 9. In some embodiments

- 59 045580 implementations TILs that exhibit granzyme B secretion greater than 19,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 20,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 40,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 60,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 80,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 100,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 120,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 140,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 160,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 180,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 200,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 220,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 240,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 260,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 280,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 300,000 pg/10 ⁶ TILs are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, TILs that exhibit granzyme B secretion of greater than 1,000 pg/ml to 300,000 pg/ml or greater are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 1000 pg/ml, greater than 2000 pg/ml, greater than 3000 pg/ml, greater than 4000 pg/ml , more than 5000 pg/ml, more than 6000 pg/ml, more than 7000 pg/ml, more than 8000 pg/ml, more than 9000 pg/ml, more than 10000 pg/ml, more than 20000 pg/ml, more than 30000 pg/ml, more 40,000 pg/ml, greater than 50,000 pg/ml, greater than 60,000 pg/ml, greater than 70,000 pg/ml, greater than 80,000 pg/ml, greater than 90,000 pg/ml, greater than 100,000 pg/ml or greater are TILs produced by propagation methods the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 1000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 2000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion are more

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3000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 4000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 5000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 6000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 7000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 8000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 9000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 10,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 20,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 30,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 40,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 50,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 60,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 70,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 80,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 90,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 100,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 120,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 140,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 160,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 180,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 200,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 220,000 pg/ml are TILs

- 61 045580 obtained by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 240,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 260,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 280,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs that exhibit granzyme B secretion greater than 300,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example, methods presented in Fig. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, the expansion methods of the present invention produce an expanded population of TILs that exhibit increased secretion of granzyme B in vitro, including, for example, the TILs shown in FIG. 2A and/or 2B and/or 2C and/or 9, compared to an unexpanded TIL population. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least one to fifty times or more compared to an unexpanded population of TILs. In some embodiments, IFN-γ secretion is increased by at least one-fold, at least two-fold, at least three-fold, at least four-fold, at least five-fold, at least six-fold, at least seven-fold , at least eight times, at least nine times, at least ten times, at least twenty times, at least thirty times, at least forty times, at least fifty times or more compared to the non-propagated TIL population. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least one-fold compared to an unexpanded population of TILs. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least twofold compared to an unexpanded population of TILs. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least three times compared to an unexpanded population of TILs. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least four times compared to an unexpanded population of TILs. In some embodiments, the secretion of granzyme B in an expanded population of TILs of the present invention is increased by at least five-fold compared to an unexpanded population of TILs. In some embodiments, the secretion of granzyme B by an expanded population of TILs of the present invention is increased by at least six-fold compared to an unexpanded population of TILs. In some embodiments, the secretion of granzyme B by an expanded population of TILs of the present invention is increased by at least seven-fold compared to an unexpanded population of TILs. In some embodiments, the granzyme secretion of an expanded population of TILs of the present invention is increased by at least eight-fold compared to an unexpanded population of TILs. In some embodiments, granzyme secretion in an expanded TIL population of the present invention is increased by at least nine-fold compared to an unexpanded TIL population. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least ten-fold compared to an unexpanded population of TILs. In some embodiments, granzyme secretion in an expanded TIL population of the present invention is increased by at least twenty-fold compared to an unexpanded TIL population. In some embodiments, granzyme B secretion is increased by at least thirty-fold in an expanded population of TILs of the present invention compared to an unexpanded population of TILs. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least forty-fold compared to an unexpanded population of TILs. In some embodiments, granzyme secretion in an expanded population of TILs of the present invention is increased by at least fifty-fold compared to an unexpanded population of TILs.

In some embodiments, phenotypic characterization is examined after cryopreservation.

J. Metabolic health of expanded TILs.

Re-stimulated TILs are characterized by a significant increase in basal glycolysis compared to freshly harvested TILs and/or post-thaw TILs. In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, a harvested TIL population, and/or a therapeutic TIL population based on CD8 expression is not performed during any of the steps including those described above or shown, for example, in FIG. 9. In some embodiments, selection of the first TIL population based on CD8 expression is not performed. In some variants

- 62 045580 selection of the second population of TILs based on CD8 expression is not performed. In some embodiments, the selection of the third TIL population based on CD8 expression is not performed. In some embodiments, selection of the harvested TIL population based on CD8 expression is not performed. In some embodiments, selection of a TIL therapeutic population based on CD8 expression is not performed.

In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, or a harvested TIL population based on CD8 expression is not performed in any of steps (a)-(f) of the method of expanding tumor infiltrating lymphocytes (TILs) into a therapeutic TIL population , including:

(a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to a closed system;

(c) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system;

(d) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third a population of TILs, wherein the third population of TILs is a therapeutic population of TILs that includes an increased subpopulation of effector T cells and/or central memory T cells compared to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system;

(e) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening the system.

In one embodiment, selecting a first TIL population, a second TIL population, a third TIL population, or a pooled TIL population based on CD8 expression and is not performed in any of steps (a) to (h) of a method of treating a subject with cancer, wherein the method includes administering expanded tumor infiltrating lymphocytes (TIL) and includes:

(a) obtaining a first population of TILs from a tumor resected from a subject by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) adding tumor fragments to a closed system;

(c) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system;

(d) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third a population of TILs, wherein the third population of TILs is a therapeutic population of TILs that includes an increased subpopulation of effector T cells and/or central memory T cells compared to the second population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system;

(e) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening;

(g) optionally cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process; and (h) administering to the patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

TILs produced by the methods described herein are characterized by a significant increase in basal glycolysis compared to, for example, freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than presented in Fig. 9. In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, a harvested TIL population, and/or a therapeutic TIL population based on CD8 expression is not performed during any of the steps including those described above or shown, for example, in FIG. . 9. In some embodiments, selection of the first TIL population based on CD8 expression is not performed. In some embodiments, selection of the second TIL population based on CD8 expression is not performed. In some embodiments, the selection of the third TIL population based on CD8 expression is not performed. In some embodiments, selection of the harvested TIL population based on CD8 expression is not performed. In some embodiments, selection of a TIL therapeutic population based on CD8 expression is not performed. In one embodiment, the selection of a first TIL population, a second TIL population, a third TIL population, or a harvested TIL population based on CD8 expression is not performed in any of steps (a)(h).

Spare respiratory capacity (SRC) and glycolytic reserve can be assessed for expanded TILs using various methods of the present invention. The Seahorse XF Cell Mito Stress Test measures mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells using respiration modulators that target components of the electron transport chain in the mitochondria. Test compounds (oligomycin, FCCP and a mixture of rotenone and antimycin A, described below) are sequentially administered to measure ATP production, maximal respiration and non-mitochondrial respiration, respectively. Proton leak and spare respiratory capacity are then calculated using these parameters and basal respiration. Each modulator targets a specific component of the electron transport chain. Oligomycin inhibits ATP synthase (complex V), and the decrease in OCR after oligomycin injection correlates with mitochondrial respiration associated with cellular ATP production. Carbonyl cyanide-4 (trifluoromethoxy)phenylhydrazone (FCCP) is an uncoupling agent that disrupts the proton gradient and disrupts mitochondrial membrane potential. As a result, the flow of electrons along the electron transport chain is not inhibited, and oxygen is maximally consumed by complex IV. The FCCP-stimulated OCR can then be used to calculate respiratory reserve capacity, defined as the difference between maximal respiration and basal respiration. Spare respiratory capacity (SRC) is a measure of a cell's ability to respond to increased energy demands. The third injection is a mixture of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor. This combination disables mitochondrial respiration and allows the calculation of non-mitochondrial respiration caused by processes outside the mitochondria. In some embodiments, comparisons are made, for example, to freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIGS. 9.

In some embodiments, the metabolic assay is basal respiration. Typically, TILs after the second breeding have a basal respiration rate that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% of the basal respiration rate of freshly collected TILs and/or TILs , obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the basal respiration rate is from about 50% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the basal respiration rate is from about 60% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the basal respiration rate is from about 70% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the basal respiration rate is from about 80% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the basal respiration rate is from about 90% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the basal respiration rate is from about 95% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs after a second propagation or TILs after a second additional propagation (such as, for example, those described in step D of FIG. 9, including TILs called reREP TILs) have a basal respiration rate that is statistically

- 64 045580 is not significantly different from the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, comparisons are made, for example, with freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9.

In some embodiments, the metabolic analysis represents spare respiratory capacity. Typically, TILs after a second propagation or TILs after a second additional propagation (such as, for example, those described in step D of Fig. 9, including TILs called reREP TILs) have a spare respiratory capacity that is at least 50%, at least at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. . 9. In some embodiments, the spare respiratory capacity is from about 50% to about 99% of the basal respiration rate of freshly collected TILs. In some embodiments, the spare respiratory capacity is from about 50% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the spare respiratory capacity is from about 60% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the spare respiratory capacity is from about 70% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the spare respiratory capacity is from about 80% to about 99% of the basal respiration rate of freshly collected TILs. In some embodiments, the spare respiratory capacity is from about 90% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the spare respiratory capacity is from about 95% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs after a second propagation or TILs after a second additional propagation (such as those described in step D of FIG. 9, including TILs called reREP TILs) have a spare respiratory capacity that is not statistically significantly different from the frequency basal respiration of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. 9.

Typically, TILs after a second propagation or TILs after a second additional propagation (such as, for example, those described in step D of Fig. 9, including TILs called reREP TILs) have a spare respiratory capacity that is at least 50%, at least at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, at least 97%, at least 98%, or at least 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods other than those described herein, including, for example, methods other than those presented in FIG. . 9. In some embodiments, the metabolic assay being measured is glycolytic reserve. In some embodiments, the metabolic analysis represents spare respiratory capacity. To measure cellular (respiratory) metabolism, cells were treated with inhibitors of mitochondrial respiration and glycolysis to determine the metabolic profile of TILs, consisting of the following parameters: baseline oxidative phosphorylation (determined by OCR), spare respiratory capacity, baseline glycolytic activity (determined by ECAR) and glycolytic reserve. Metabolic profiles were obtained using the Seahorse combined mitochondrial/glycolysis stress test (including a kit commercially available from Agilent®), which measures the ability of cells to perform glycolysis while blocking mitochondrial ATP production. In some embodiments, the cells are glucose starved, then administered glucose and then a stress agent. In some embodiments, the stress agent is selected from the group consisting of oligomycin, FCCP, rotenone, antimycin A, and/or 2-deoxyglucose (2-DG), as well as combinations thereof. In some embodiments, oligomycin is added in an amount of 10 mM. In some embodiments, FCCP is added in an amount of 10 mM. In some embodiments, rotenone is added in an amount of 2.5 mM. In some embodiments, antimycin A is added in an amount of 2.5 mM. In some embodiments, 2-deoxyglucose

- 65 045580 (2-DG) is added in an amount of 500 mM. In some embodiments, glycolytic capacity, glycolytic reserve, and/or non-glycolytic acidification are measured. Typically, TILs have a glycolytic reserve that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods different from those described herein, including, for example, methods other than those presented in FIGS. 9. In some embodiments, the glycolytic reserve is from about 50% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 60% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 70% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 80% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 90% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs prepared by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 95% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9.

In some embodiments, the metabolic assay is basal glycolysis. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as, for example, those described in step D of Fig. 9, including TILs called reREP TILs) have an increase in basal glycolysis of at least twofold, at least three times, at least four times, at least five times, at least six times, at least seven times, at least seven times, at least nine times, at least ten times compared to freshly collected TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as those described in step D of FIG. 9, including TILs called reREP TILs) have about a two- to about ten-fold increase in basal glycolysis compared to freshly harvested TIL and/or TIL produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as those described in step D of FIG. 9, including TILs called reREP TILs) have about a two- to about eight-fold increase in basal glycolysis compared to freshly harvested TIL and/or TIL produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as those described in step D of FIG. 9, including TILs called reREP TILs) have about a two- to about seven-fold increase in basal glycolysis compared to freshly harvested TIL and/or TIL produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as those described in step D of FIG. 9, including TILs called reREP TILs) have an increase in basal glycolysis of about two to about four times per compared to freshly harvested TIL and/or TIL produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as those described in step D of FIG. 9, including TILs called reREP TILs) have about a two- to about three-fold increase in basal glycolysis compared to freshly harvested TIL and/or TIL produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9.

Typically, TILs after a second expansion or TILs after a second additional expansion (such as, for example, those described in step D in Fig. 9, including TILs called reREP TILs) have a glycolytic reserve that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, according to

- 66 045580 at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% of the basal respiration rate of freshly collected TILs and/or TILs obtained by methods, different from those described herein, including, for example, methods other than those presented in FIGS. 9. In some embodiments, the glycolytic reserve is from about 50% to about 99% of the basal respiration rate of freshly collected TILs. In some embodiments, the glycolytic reserve is from about 60% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 70% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 80% to about 99% of the basal respiration rate of freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 90% to about 99% of the basal respiration rate of freshly collected TILs and/or TILs prepared by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, the glycolytic reserve is from about 95% to about 99% of the basal respiration rate of freshly collected TILs.

Granzyme B production Granzyme B is another indicator of the ability of TILs to kill target cells. Medium supernatants restimulated as described above with antibodies to CD3, CD28, and CD137/4-1BB were also assessed for granzyme B content using the Human Granzyme B DuoSet ELISA kit (R&D Systems, Minneapolis, MN). in accordance with the manufacturer's instructions. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as those described in step D of FIG. 9, including TILs called reREP TILs) increase granzyme B production. In some embodiments, TILs after a second expansion or TILs after a second additional expansion (such as those described in step D in FIG. 9, including TILs called reREP TILs) have increased cytotoxic activity.

In some embodiments, telomere length can be used as a measure of cell viability and/or cellular function. In some embodiments, telomeres are surprisingly the same length in TILs produced by methods of the present invention compared to TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. Telomere length measurement: Various methods have been used to measure telomere length in genomic DNA and cytological preparations. Telomere restriction fragment (TRF) analysis is the gold standard for measuring telomere length (de Lange et al., 1990). However, the main limitation of the TRF assay is the large amount of DNA required (1.5 μg). Two commonly used methods for measuring telomere length, namely fluorescence in situ hybridization (FISH; Agilent Technologies, Santa Clara, California) and quantitative PCR, can be used with the present invention. In some embodiments, there is no change in telomere length between the initially assembled TILs in step A and the expanded TILs, for example, in step D, as shown in FIG. 9.

In some embodiments, the TILs express one or more markers selected from the group consisting of granzyme B, perforin, and granulysin. In some embodiments, the TILs express granzyme B. In some embodiments, the TILs express perforin. In some embodiments, the TILs express granulysin.

In one embodiment, re-stimulated TILs can also be assessed for cytokine release using cytokine release assays. In some embodiments, TILs can be assessed for interferon-γ (IFN-γ) secretion. In some embodiments, IFN-γ secretion is measured using an ELISA assay. In some embodiments, IFN-γ secretion is measured using an ELISA assay after the rapid second expansion step, after step D, as shown, for example, in FIG. 2 (in particular, for example, in Fig. 2A and/or 2B, and/or 2C, and/or 9). In some embodiments, TIL health is measured by the secretion of interferon gamma (IFN-γ). In some embodiments, IFN-γ secretion is an indicator of the presence of active TILs. In some embodiments, an efficacy assay based on determining IFN-γ production is used. IFN-γ production is another indicator of cytotoxic potential. IFN-γ production can be measured by determining the levels of the cytokine IFN-γ in TIL media stimulated with antibodies to CD3, CD28 and CD137/4-1BB. The levels of IFN-γ in the media from these stimulated TILs can be determined by measuring the release of IFN-γ. In some embodiments, increasing IFN-γ production, for example, in step D of the Gen 3 process, as shown in FIG. 2 (in particular, for example, Fig. 2A and/or 2B, and/or 2C, and/or 9) TIL compared, for example, with step D in

- 67 045580 process 2A, as shown in FIG. 2 (particularly, e.g., FIG. 2A) indicates an increase in the cytotoxic potential of TILs during step D. In some embodiments, IFN-γ secretion is increased by one-fold, two-fold, three-fold, four-fold, or five-fold or more. In some embodiments, IFN-γ secretion is increased by one-fold. In some embodiments, IFN-γ secretion is increased twofold. In some embodiments, IFN-γ secretion is increased threefold. In some embodiments, IFN-γ secretion is increased fourfold. In some embodiments, IFN-γ secretion is increased fivefold. In some embodiments, the level of IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, the level of IFN-γ is measured in TILs ex vivo. In some embodiments, the level of IFN-γ is measured in TILs ex vivo, including TILs produced by the methods of the present invention, including, for example, the methods presented in FIG. 2B.

In some embodiments, TILs capable of increasing IFN-γ secretion by at least one-fold, two-fold, three-fold, four-fold, five-fold, or more are TILs produced by the propagation methods of the present invention, including, for example, methods , presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of increasing IFN-γ secretion by at least one-fold are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of increasing IFN-γ secretion by at least twofold are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of increasing IFN-γ secretion by at least threefold are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of increasing IFN-γ secretion by at least fourfold are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of increasing IFN-γ secretion by at least five-fold are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, TILs capable of secreting IFN-γ from at least 100 pg/ml to about 1000 pg/ml or more are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 200 pg/ml, at least 250 pg/ml, at least 300 pg/ml ml, at least 350 pg/ml, at least 400 pg/ml, at least 450 pg/ml, at least 500 pg/ml, at least 550 pg/ml, at least 600 pg/ml , at least 650 pg/ml, at least 700 pg/ml, at least 750 pg/ml, at least 800 pg/ml, at least 850 pg/ml, at least 900 pg/ml, according at least 950 pg/ml or at least 1000 pg/ml or more are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 200 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 200 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 300 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 400 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 500 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 600 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 700 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 800 pg/ml are TILs produced by the methods

- 68 045580 propagation according to the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 900 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 1000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 2000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 3000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 4000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 5000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 6000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 7000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 8000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 9000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 10,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 15,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 20,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 25,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 30,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 35,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 40,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 45,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 50,000 pg/ml are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, TILs capable of secreting IFN-γ from at least 100 pg/ml/5e5 cells to about 1000 pg/ml/5e5 cells or more are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 200 pg/ml/5e5 cells, at least 250 pg/ml/5e5 cells, at least 300 pg/ml/5e5 cells, at least 350 pg/ml/5e5 cells, at least 400 pg/ml/5e5 cells, by

- 69 045580 at least 450 pg/ml/5e5 cells, at least 500 pg/ml/5e5 cells, at least 550 pg/ml/5e5 cells, at least 600 pg/ml/5e5 cells, at least 650 pg/ml/5e5 cells, at least 700 pg/ml/5e5 cells, at least 750 pg/ml/5e5 cells, at least 800 pg/ml/5e5 cells, at least 850 pg/ml/5e5 cells at least 900 pg/ml/5e5 cells, at least 950 pg/ml/5e5 cells, or at least 1000 pg/ml/5e5 cells or more are TILs produced by the propagation methods of the present invention, including, for example , the methods presented in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 200 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 200 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 300 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 400 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 500 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 600 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 700 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 800 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 900 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 1000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 2000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 3000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 4000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 5000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 6000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 7000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 8000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 9000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 10,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, ways to represent

- 70 045580 lined in Fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 15,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 20,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 25,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 30,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 35,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ of at least 40,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 45,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting IFN-γ at least 50,000 pg/ml/5e5 cells are TILs produced by the propagation methods of the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, TILs capable of reducing the level of TNF-α secretion (i.e., TNF-alpha) by at least one-fold, two-fold, three-fold, four-fold, five-fold, or more compared to IFN-γ secretion , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of reducing the level of TNF-α secretion relative to IFN-γ secretion by at least one-fold are TILs produced by the methods propagation according to the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of reducing the level of TNFα secretion relative to IFN-γ secretion by at least two-fold are TILs produced by propagation methods the present invention, including, for example, the methods shown in FIGS. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of reducing the level of TNF-α secretion relative to IFN-γ secretion by at least three times are TILs produced by the methods propagation according to the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of reducing the level of TNF-α secretion relative to IFN-γ secretion by at least four times are TILs produced by the methods propagation according to the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of reducing the level of TNF-α secretion relative to IFN-γ secretion by at least five times are TILs produced by the methods propagation according to the present invention, including, for example, the methods presented in FIG. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, TILs capable of secreting TNF-α (i.e., TNF-alpha) from at least 200 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more are TILs produced by the methods propagation according to the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 500 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 1000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 2000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 3000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including,

- 71 045580 for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 4,000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 5,000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 6,000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 7,000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 8,000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, TILs capable of secreting TNF-α from at least 9,000 pg/ml/5e5 cells to about 10,000 pg/ml/5e5 cells or more , are TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, IFN-γ and granzyme B levels are measured to determine the phenotypic characteristics of TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. 2A and/or 2B and/or 2C and/or 9. In some embodiments, IFN-γ and TNF-α levels are measured to determine the phenotypic characteristics of TILs produced by the propagation methods of the present invention, including, for example, the methods presented in fig. 2A and/or 2B and/or 2C and/or 9. In some embodiments, levels of granzyme B and TNF-α are measured to determine the phenotypic characteristics of TILs produced by the propagation methods of the present invention, including, for example, the methods presented in FIG. . 2A and/or 2B and/or 2C and/or 9. In some embodiments, levels of IFN-γ, granzyme B, and TNF-α are measured to determine the phenotypic characteristics of TILs produced by propagation methods of the present invention, including, for example, methods , presented in Fig. 2A and/or 2B, and/or 2C, and/or 9.

In some embodiments, the cytotoxic potential of TILs to lyse target cells is assessed using a TIL coculture assay with the bioluminescent cell line P815 (clone G6) according to a bioluminescent redirected lysis assay (activity assay) for a TIL assay that measures TIL cytotoxicity in a highly sensitive, dose-dependent manner. way.

In some embodiments, the present methods provide an assay to assess the viability of TILs in the manner described above. In some embodiments, TILs are propagated as described above, including, for example, as shown in FIG. 9. In some embodiments, TILs are cryopreserved before their viability is assessed. In some embodiments, the viability assessment includes thawing the TIL before performing a first expansion, a second expansion, and an additional second expansion. In some embodiments, the present methods provide an assay to assess cell proliferation, cell toxicity, cell death, and/or other conditions associated with the viability of a TIL population. Viability can be measured using any of the TIL metabolic assays described above, as well as any methods known in the art to assess cell viability. In some embodiments, the present methods provide an assay to assess cell proliferation, cell toxicity, cell death, and/or other conditions associated with the viability of TILs propagated by the methods described herein, including the methods presented in FIG. 9.

The present invention also provides assay methods for determining the viability of TILs. In some embodiments, TILs have equal viability compared to freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs have increased viability compared to freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. The present invention provides methods for assaying TILs for viability by expanding tumor infiltrating lymphocytes (TILs) into a larger population of TILs, comprising:

- 72 045580 (i) obtaining a first TIL population that has been previously propagated;

(ii) performing a first propagation by culturing a first population of TILs in a cell culture medium containing IL-2 to obtain a second population of TILs;

(iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the third population of TILs is at least 100 times larger than the second population of TILs and wherein the second expansion is performed for at least 14 days to obtain a third population of TILs, wherein the third population of TILs includes an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs, and wherein the third population is further assayed for viability.

In some embodiments of the present invention, the method further includes:

(iv) performing an additional second expansion by adding additional IL-2, additional OKT-3, and additional APCs to the cell culture medium of the third TIL population, wherein the additional second expansion is performed over at least 14 days to obtain a larger TIL population than that obtained in step (iii), wherein the larger TIL population includes an increased subpopulation of effector T cells and/or central memory T cells compared to the third TIL population, and wherein the third population is further assayed for viability.

In some embodiments, the cells are cryopreserved prior to step (i).

In some embodiments, the cells are thawed before step (i) is performed.

In some embodiments, step (iv) is repeated one to four times to obtain a sufficient amount of TIL for analysis.

In some embodiments, steps (i)-(iii) or (iv) are performed over a period of about 40 days to about 50 days.

In some embodiments, steps (i)-(iii) or (iv) are performed over a period of about 42 days to about 48 days.

In some embodiments, steps (i)-(iii) or (iv) are performed over a period of about 42 days to about 45 days.

In some embodiments, steps (i)-(iii) or (iv) are performed over a period of about 44 days.

In some embodiments, cells from steps (iii) or (iv) express CD4, CD8, and TCR αβ at levels similar to freshly harvested cells.

In some embodiments, the antigen presenting cells are peripheral blood mononuclear cells (PBMCs).

In some embodiments, PBMCs are added to the cell culture on any of days 9 to 17 in step (iii).

In some embodiments, effector T cells and/or central memory T cells in the larger population of TILs in step (iv) exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression, longer telomeres, increased CD57 expression and decreased expression of CD56 compared to effector T cells and/or central memory T cells in a third cell population.

In some embodiments, effector T cells and/or central memory T cells exhibit increased expression of CD57 and decreased expression of CD56.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the method further includes the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the method further includes the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the TILs are assayed for viability.

In some embodiments, TILs are assayed for viability after cryopreservation.

In some embodiments, TILs are analyzed for viability after cryopreservation and after step (iv).

A variety of antigen receptors of T and B lymphocytes are formed as a result of somatic recombination of a limited but large number of gene segments. These gene segments include: V (variability), D (diversity), J (attachment) and C (constant) determine the binding specificity and subsequent applications of immunoglobulins and T-cell receptors (TCRs). The present invention provides a method for generating TILs that exhibit and enhance T cell repertoire diversity (sometimes referred to as polyclonality). In some embodiments, the increase in T cell repertoire diversity occurs relative to freshly harvested TILs and/or TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIG. 9. In some embodiments, TILs produced by the present method exhibit increased T cell repertoire diversity. In some embodiments, the TILs obtained from the first expansion exhibit an increase in T cell repertoire diversity. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is present in the immunoglobulin heavy chain. In some embodiments, the immunoglobulin variety is present in the immunoglobulin light chain. In some embodiments, diversity is present in the T cell receptor. In some embodiments, the diversity is present in one of the T cell receptors selected from the group consisting of alpha, beta, gamma, and delta receptors. In some embodiments, an increase in T cell receptor (TCR) alpha and/or beta expression is observed. In some embodiments, an increase in T cell receptor (TCR) alpha expression is observed. In some embodiments, an increase in T cell receptor (TCR) beta expression is observed. In some embodiments, an increase in TCRab (ie, TCRa/β) expression is observed.

The present invention provides a method for analyzing TILs for viability and/or further use when administered to a subject. In some embodiments, a method for analyzing tumor infiltrating lymphocytes (TILs) includes:

(i) obtaining the first TIL population;

(ii) performing a first propagation by culturing a first population of TILs in a cell culture medium containing IL-2 to obtain a second population of TILs;

(iii) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the third population of TILs is at least 50 times larger than the second population of TILs ;

(iv) collecting, washing and cryopreserving the third population of TILs;

(v) storing cryopreserved TILs at cryogenic temperatures;

(vi) thawing the third TIL population to obtain a thawed third TIL population;

(vii) performing an additional second expansion of a portion of the thawed third population of TILs by adding IL-2, OKT-3, and APC to the cell culture medium of the third population for an additional expansion period (sometimes referred to as a reREP period) of at least 3 days, wherein a third propagation is performed to obtain a fourth TIL population, the number of TILs in the fourth TIL population being compared with the number of TILs in the third TIL population to obtain a ratio;

(viii) determining, based on the ratio obtained in step (vii), the suitability of the thawed TIL population for administration to a patient;

(ix) administering to the patient a therapeutically effective dose of the thawed third TIL population when the ratio of the amount of TIL in the fourth TIL population to the amount of TIL in the third TIL population is determined to be greater than 5:1 in step (viii).

In some embodiments, an additional propagation period (sometimes referred to as a reREP period) is performed until the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is greater than 50:1.

In some embodiments, the amount of TIL sufficient for a therapeutically effective dose is from about 2.3 x 10 ¹⁰ to about 13.7 x 10 1°.

In some embodiments, steps (i)-(vii) are performed over a period of about 40 days to about 50 days. In some embodiments, steps (i)-(vii) are performed over a period of from about 42 days to about 48 days. In some embodiments, steps (i)-(vii) are performed over a period of from about 42 days to about 45 days. In some embodiments, steps (i)-(vii) are performed over a period of about 44 days.

In some embodiments, cells from steps (iii) or (vii) express CD4, CD8, and TCR αβ at levels similar to freshly harvested cells. In some embodiments, the cells are TILs.

In some embodiments, the antigen presenting cells are peripheral blood mononuclear cells (PBMCs). In some embodiments, PBMCs are added to the cell culture on any of days 9 to 17 in step (iii).

In some embodiments, effector T cells and/or central memory T cells in the larger TIL population at steps (iii) or (vii) exhibit one or more characteristics selected from the group consisting of CD27 expression, CD28 expression , longer telomeres, increased CD57 expression and decreased CD56 expression compared to effector T cells and/or central memory T cells in the third cell population.

In some embodiments, effector T cells and/or central memory T cells exhibit increased expression of CD57 and decreased expression of CD56.

In some embodiments, the APCs are artificial APCs (aAPCs).

In some embodiments, the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor is performed.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) containing a single chain variable fragment antibody fused to at least one endodomain of a T cell signaling molecule is performed.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the TILs are assayed for viability after step (vii).

The present invention also provides additional methods for analyzing TILs. In some embodiments, the present invention provides a method for analyzing TILs, comprising:

(i) obtaining a portion of the first population of cryopreserved TILs;

(ii) thawing a portion of the first population of cryopreserved TILs;

(iii) performing a first expansion by culturing a portion of the first TIL population in a cell culture medium containing IL-2, OKT-3, and antigen presenting cells (APCs) for an additional expansion period (sometimes called a reREP period) of at least 3 day, producing a second population of TILs, wherein a portion of the first TIL population is compared with a second TIL population to obtain a ratio of the number of TILs, wherein the ratio of the number of TILs in the second TIL population to the number of TILs in a portion of the first TIL population is greater than 5:1;

(iv) determining, based on the ratio obtained in step (iii), the suitability of the first population of TILs for use in therapeutic administration to a patient;

(v) determining the suitability of the first population of TILs for use in therapeutic administration when the ratio of the amount of TILs in the second population of TILs to the amount of TILs in the first population of TILs is determined to be greater than 5:1 in step (iv).

In some embodiments, the ratio of the number of TILs in the second TIL population to the number of TILs in a portion of the first TIL population is greater than 50:1.

In some embodiments, the method further includes performing propagation of the entire first population of cryopreserved TILs from step (i) in accordance with the methods described in any of the embodiments presented herein.

In some embodiments, the method further includes administering to the patient the entire first population of cryopreserved TILs from step (i).

K. Closed systems for TIL production

The present invention relates to the use of closed systems in the process of culturing TILs. Such closed systems prevent and/or reduce microbial contamination, allow the use of fewer flasks, and reduce costs. In some embodiments, two containers are used in a closed system.

Such closed systems are well known in the art and their description can be found, for example, at http://www.fda.gov/cber/guidelines.htm and https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances /Blood/ucm076779.htm.

As stated on the FDA website, closed sterile culture systems and methods are known and described in detail, see https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Blood/ucm076779.htm, above, and the description of the relevant part below.

Introduction.

Sterile Coupling Devices (STCDs) create sterile welds between two pieces of compatible tubing. This procedure allows for the sterile connection of containers and pipes of different diameters. This manual describes the recommended methods and procedures for using these devices. This guidance does not address information about the data that a manufacturer of a sterile connecting device must submit to the FDA to obtain approval or marketing authorization. It is also important to note that use of an approved or marketed sterile connecting device for purposes other than those specified in the data sheet may result in the device being considered adulterated and mislabeled under the Federal Food, Drug, and Fragrance Act. cosmetic products.

1. FDA recommendations.

Manufacturers of blood products planning to routinely use FDA-approved STCDs should include information about such use in the standard operating procedure (SOP) for

- 75 045580 for each blood product. These records should include inventory, product tracking, weld quality control, software lot numbers, and discarded materials (including source(s) of added items). Quality control procedures should include checking the integrity of each weld.

2. Application of STCD.

The user should be aware that use of the device may result in the creation of a new product, or a significant change in the configuration of a regulated product, for which safety and effectiveness have not been demonstrated. For those new products that are subject to licensure, applications, or supplements to applications, must be filed with the FDA, in addition to filing an SOP. Generally, combining or mixing cellular components constitutes a change in the product that requires the submission and approval of an application, or amendment to the application, for licensing. Such applications, or application supplements, must contain data and descriptions of manufacturing procedures that demonstrate that the new product is safe and effective for its intended use during the proposed period.

The following comments are provided as a guide to the more common uses of FDA-approved or approved STCDs.

L. Adding a new or smaller needle to the blood collection kit.

Using an STCD to add a needle prior to the start of a procedure (whole blood collection, thrombopheresis, or plasma source collection) is not considered to be opening a functionally closed system. If a needle is added during a procedure, only STCD approved for welding fluid-filled tubing should be used. If the weld integrity test is satisfactory, the use of STCD is not considered to be opening a functionally closed system.

Platelets obtained by plasmapheresis in an open system should have a specified shelf life of 24 hours, and platelets obtained by plasmapheresis in a functionally closed system should have a specified shelf life of five days (see Revised Guideline for Collection of Platelets, Pheresis, October 7, 1988).

The source and specifications of added tubing and needles should be noted in the SOP and blood collection center records. The use of STCD to add needles is not a major change in the manufacturing process for which licensed establishments must obtain prior approval.

M. Application of STCD for preparation of components.

When STCD is used to attach additional bags for component preparation, proper records should be maintained indicating the source of the transfer bags and appropriate verification of the number of blood units and ABO/Rh. All blood and blood components must be labeled as such (21 CFR 606.121).

Examples

Adding a fourth bag to a triple whole blood collection bag to produce AHF cryoprecipitate from fresh frozen plasma.

Connecting additional solution to a unit of packed red blood cells.

Adds an inline filter that has been cleared by the FDA for use in component manufacturing.

Adding a third storage container to the thrombopheresis kit.

Procedures and records should be maintained for the above uses, but licensees are not required to obtain FDA approval to perform procedures.

1. Use of STCD for pooling blood products Appropriate use of STCD for pooling platelets derived from whole blood can eliminate potential contamination from commonly used needles and ports. Combination performed immediately prior to transfusion is an example of such a suitable application. Pooled platelets should be administered no more than 4 hours after pooling (see 21 CFR 606.122(1)(2)).

However, pooling and subsequent storage may increase the risk compared with administering platelet units from individual donors; if one contaminated unit is combined with others and stored until administration, the total bacterial inoculum administered may increase by replicating to an additional extent. Accordingly, the proposed use of STCD for pooling and storing platelets for more than 4 hours should be supported by data from an appropriate study of whether such pooling is associated with an increased risk.

This combination of platelets represents the production of a new product.

Combining or mixing platelets is considered to be the manufacture of a new product, requiring the submission and approval of an application, or amendment to an application, for a license if the shelf life exceeds four hours.

2. Use of STCD to obtain pediatric aliquots and split units.

Pediatric units and divided units for whole blood, red blood cells and

- 76 045580 fresh frozen plasma obtained through STCD will not be considered a new product requiring a Biologics Licensing Application (BLA) supplement, provided the following conditions are met:

The manufacturer must have an approved biologics license, or supplement to a license, for the originator (ie, undivided) product, including approval for each anticoagulant used.

Labels must be submitted for review and approval prior to distribution. The notation should be in the comment section of FDA Form 2567, Transmittal of Labels and Circulars.

End product containers approved for storing the component being manufactured should be used.

Platelets manufactured under the license must contain at least 5.5 x 101° platelets (21 CFR 640.24 (c)). Platelets obtained by licensed plasmapheresis must contain at least 3.0 x 1ο ¹¹ platelets (see Revised Guideline for the Collection of Platelets, Pheresis, October 7, 1988).

The procedures to be followed for the use of STCD for the preparation of separated products from whole blood and from plasma and platelets obtained using automated hemapheresis procedures must include descriptions of how the apheresis device or collection container will be modified by an approved FDA STCD;

minimum volume of separated plasma or whole blood products;

volume and concentration of platelets in the separated thrombopheresis products;

shelf life of the product. The product must be in an approved container and must comply with the shelf life stated on the label of such container;

method(s) used to label and track separated products in blood collection center records.

Note. Methods for labeling aliquots should be clearly stated in the record keeping procedure and should be sufficient to allow traceability and recall of all components, if necessary.

3. Use of STCD to connect additional saline or anticoagulant lines during an automated plasmapheresis procedure.

Procedures should be developed and records maintained that are consistent with the device manufacturer's instructions for use, but licensees are not required to have FDA approval to perform procedures.

4. Use of STCD for supplying treatment solutions.

When STCD is used to attach containers of processing solutions for washing or freezing red blood cells, the shelf life of the resulting products is 24 hours unless data is provided on the CBER license application form or application form in favor of a longer period of time ( 21 CFR 610.53(c)). Exceptions or modifications must be approved in writing by the Director, CBER (21 CFR 610.53(d)).

5. Use of STCD to supplement an FDA-approved filter to reduce white blood cell counts

In some cases, leukocyte count-reducing filters are not initially installed in whole blood collection systems. Procedures for using STCD for filtration prior to storage must comply with the filter manufacturer's instructions for use.

Reducing the white blood cell count before release is a major change to the manufacturing process. Therefore, for new STCD-derived white blood cell count products, manufacturers must submit applications, or supplements to previously approved applications, for a biologics license (21 CFR 601.2) to the FDA (21 CFR 601.12).

Use of an STCD to retrieve specimens for testing from blood product containers (eg, use of an STCD to obtain a platelet sample from a container of platelets or plasmapheresis platelets for cross-matching).

If the volume and/or cell count of a product after sampling differs from what is stated on the original label or information circular, the product label should be amended to reflect the new volume and/or cell count. For example, specimens that would reduce the platelet count per platelet mass unit to less than 5.5x(10) ¹⁰ platelets (21 CFR 640.24(c)) should not be removed.

6. Additional information from FDA guidance.

The FDA Guidance provides general guidance as well as specific information and examples related to FDA application specifications and application annexes related to the use of STCDs. If further questions arise regarding the appropriate use of STCD, inquiries should be directed to the Office of Blood Research and Review, Center for Biologics Evaluation and Research.

- 77 045580

In some embodiments, a single container is used in a closed system from the time tumor fragments are obtained until the TILs are ready for administration to a patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G-container, and the TIL population is centrifuged and transferred to an infusion bag without opening the first closed G-container. In some embodiments, when two containers are used, the infusion bag is a HypoThermosol-containing infusion bag. A closed system, or closed system for culturing TILs, is characterized in that, after the addition of a tumor sample and/or tumor fragments, the system is hermetically sealed from the outside, forming a closed environment free from invading bacteria, fungi and/or any other microbial contaminants.

In some embodiments, the degree of microbial contamination reduction is from about 5% to about 100%. In some embodiments, the degree of microbial contamination reduction is from about 5% to about 95%. In some embodiments, the degree of microbial contamination reduction is from about 5% to about 90%. In some embodiments, the degree of microbial contamination reduction is from about 10% to about 90%. In some embodiments, the degree of microbial contamination reduction is from about 15% to about 85%. In some embodiments, the degree of microbial contamination reduction is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100% .

The closed system allows TIL to grow in the absence and/or greatly reduced microbial contamination.

In addition, the pH, partial pressure of carbon dioxide and oxygen in the TIL culture medium vary during cell culture. Therefore, even though a medium suitable for cell culture is circulated, the environment in a closed system still needs to be constantly maintained in an optimal state for TIL proliferation. To do this, it is desirable to control the physical factors of pH, partial pressure of carbon dioxide and partial pressure of oxygen in the culture liquid of the closed system using a sensor, a signal that is used to control the gas exchanger installed at the entrance to the cultivation system, to adjust in real time the partial pressure of gas in the closed system. system according to changes in the culture fluid in order to optimize the cell culture environment. In some embodiments, the present invention relates to a closed cell culture system having, at the inlet of the closed system, a gas exchanger equipped with a control device that measures the pH, carbon dioxide partial pressure, and oxygen partial pressure of the closed system and optimizes the cell culture environment by automatically adjusting the concentrations gases based on signals from the control device.

In some embodiments, the pressure in the closed system is continuously or periodically monitored. That is, the pressure in a closed system can be varied by a pressure maintaining device, for example, thereby making the space suitable for TIL growth under positive pressure conditions or stimulating fluid exudation under negative pressure conditions and thus stimulating cell proliferation. Additionally, by periodically applying negative pressure, circulating fluid in a closed system can be uniformly and efficiently replaced by temporarily reducing the volume of the enclosed space.

In some embodiments, optimal culture components required for TIL proliferation may be replaced or added, including factors such as IL2 and/or OKT3, or a combination thereof, may be added.

N. Cell cultures.

In one embodiment, a method for propagating TILs including those described above and also illustrated in FIG. 9 may include the use of about 5,000 ml to about 25,000 ml of cell culture medium, from about 5,000 ml to about 10,000 ml of cell culture medium, or from about 5,800 ml to about 8,700 ml of cell culture medium. In some embodiments, the medium is a serum-free medium, such as described in Example 21. In some embodiments, the medium is serum-free when first propagated. In some embodiments, the second propagation medium does not contain serum. In some embodiments, both the first propagation medium and the second propagation medium do not contain serum. In one embodiment, no more than one type of cell culture medium is used to propagate TILs. Any suitable cell culture medium can be used, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, Calif.). In this regard, the patented methods have the advantage of reducing the amount of media, as well as the number of types of media required for TIL propagation. In one embodiment, TIL propagation may involve feeding the cells no more frequently than every third or fourth day. Propagating cells in a gas-permeable container simplifies the procedures required for cell propagation by reducing the frequency of feeding required for cell propagation.

In an embodiment, the cell culture medium in the first and/or second gas-permeable container is unfiltered. The use of unfiltered cell media can simplify the procedures required for cell propagation. In one embodiment, beta-mercaptoethanol (BME) is absent from the cell environment in the first and/or second gas-permeable container.

In one embodiment, the duration of the method includes obtaining a sample of tumor tissue from a mammal; culturing a sample of tumor tissue in a first gas-permeable container containing a cell medium; obtaining TIL from a tumor tissue sample; propagating the TILs in a second gas-permeable container containing cell media for a period of about 7 to about 14 days, such as about 11 days. In some embodiments, the pre-REP takes about 7-14 days, such as about 11 days. In some embodiments, the REP takes about 714 days, such as about 11 days.

In one embodiment, TILs are propagated in gas-permeable containers. Gas-permeable containers have been used to propagate TILs using PBMCs using methods, compositions and devices known in the art, including those described in US Patent Application Publication No. 2005/0106717 A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are propagated in gas-permeable bags. In one embodiment, TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are propagated using a cell expansion system that propagates TILs in gas-permeable bags, such as the WAVE bioreactor system, also known as the Xuri W5 cell expansion system (GE Healthcare). In one embodiment, the cell propagation system includes a gas-permeable cell bag with a volume selected from the group consisting of about 100 ml, about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml, about 700 ml, about 800 ml, about 900 ml, about 1 l, about 2 l, about 3 l, about 4 l, about 5 l, about 6 l, about 7 l, about 8 l, about 9 l and about 10 l.

In one embodiment, TILs can be propagated in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such embodiments allow the cell population to be expanded from about 5x105 cells/ ^cm2 to 10x106 to 30x106 cells/ ^cm2 . In an embodiment, this occurs without replenishment. In one embodiment, this occurs without feeding while the medium is at a height of about 10 cm in the G-Rex flask. In one embodiment, this occurs without feeding, but with the addition of one or more cytokines. In one embodiment, the cytokine can be added as a bolus without the need to mix the cytokine with the medium. Such containers, devices and methods are known in the art and have been used for propagating TILs, including those described in US Patent Application Publication No. US 2014/0377739A1, International Publication No. WO 2014/210036 A1, US Patent Application Publication No. US 2013/0115617 A1, International Publication No. WO 2013/188427 A1, US Patent Application Publication No. US 2011/0136228 A1, US Patent No. US 8809050 B2, International Publication No. WO 2011/072088 A2, US Patent Application Publication No. US 2016/0208216 A1 , US Patent Application Publication No. US 2012/0244133 A1, International Publication No. WO 2012/129201 A1, US Patent Application Publication No. US 2013/0102075 A1, US Patent Application Publication No. US 8956860 B2, International Publication No. WO 2013/173835 A1, US Patent Application Publication No. US 2015/0175966 A1, the disclosure of which is incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

A. Optional genetic modification of TILs.

In some embodiments, TILs are optionally genetically modified to include additional functional properties, including, but not limited to, a high-affinity T cell receptor (TCR), e.g., a TCR targeting a tumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen receptor (CAR) that binds to a tumor-associated cell surface molecule (eg, mesothelin) or a lineage-specific cell surface molecule (eg, CD 19).

R. Optional cryopreservation of TILs.

Either the total TIL population or the expanded TIL population can optionally be cryopreserved. In some embodiments, a therapeutic population of TILs is cryopreserved. In some embodiments, TILs collected after the second expansion are cryopreserved. In some embodiments, TILs are cryopreserved in exemplary step F shown in FIG. 9. In some embodiments, the TIL is cryopreserved in an infusion bag. In some embodiments, the TIL is cryopreserved prior to placement in the infusion bag. In some embodiments, the TILs are cryopreserved and not placed in an infusion bag. In some embodiments, cryopreservation is performed using a cryopreservation medium. In some embodiments, the cryopreservation medium contains dimethyl sulfoxide

- 79 045580 (DMSO). Cryopreservation is typically accomplished by placing a population of TILs in a freezing solution, for example, containing 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). Cells in solution are placed in cryogenic vials and stored for 24 hours at 80°C with optional transfer to nitrogen gas freezers for cryopreservation, see Sadeghi, et al., Acta Oncologica 2013, 52, 978-986.

If necessary, the cells are removed from the freezer and thawed at 37°C in a water bath until approximately 4/5 of the solution is thawed. Cells are typically resuspended in complete medium and optionally washed one or more times. In some embodiments, thawed TILs can be counted and assessed for viability, as is known in the art.

In a preferred embodiment, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLife Solutions). In a preferred embodiment, the TIL population is cryopreserved using a cryopreservation medium containing dimethyl sulfoxide (DMSO). In a preferred embodiment, the TIL population is cryopreserved using a 1:1 (v/v) mixture of CS10 and cell culture medium. In a preferred embodiment, the TIL population is cryopreserved with a mixture of about 1:1 (v/v) CS10 and cell culture medium further containing IL-2.

As described above for steps A-E, cryopreservation can be performed at various points throughout the TIL propagation process. In some embodiments, the population of total TILs after the first expansion in step B or the expanded population of TILs after one or more second expansions in step D can be cryopreserved. Cryopreservation can generally be accomplished by placing the TIL population in a freezing solution, for example, containing 85% AB serum with inactivated complement and 15% dimethyl sulfoxide (DMSO). Cells in solution are placed in cryogenic vials and stored for 24 hours at -80°C with optional transfer to nitrogen gas freezers for cryopreservation, see Sadeghi, et al., Acta Oncologica 2013, 52, 978-986.

If necessary, the cells are removed from the freezer and thawed at 37°C in a water bath until approximately 4/5 of the solution is thawed. Cells are typically resuspended in complete medium and optionally washed one or more times. In some embodiments, thawed TILs can be counted and assessed for viability, as is known in the art.

In some cases, the TIL population obtained in step B can be cryopreserved immediately using the protocols described below. Alternatively, the population of total TILs can be subjected to stage C and stage D, and then cryopreserved after stage D. Similarly, in the case where genetically modified TILs will be used in the therapy, TIL populations obtained from stage B or stage D can be genetically modified to appropriate treatment.

Q. Optional Cell Viability Assays.

Optionally, a cell viability assay can be performed after the first expansion (sometimes referred to as the initial bulk expansion) using standard assays known in the art. For example, a trypan blue assay can be performed on a sample of total TILs, which selectively labels dead cells and allows for assessment of viability. Other viability testing assays may include, but are not limited to, the Alamar Blue assay and the MTT assay.

1. Cell count, viability, flow cytometry.

In some embodiments, cell number and/or viability are measured. The expression of markers, such as, but not limited to, CD3, CD4, CD8 and CD56, as well as any others disclosed or described herein, can be measured by flow cytometry with antibodies, such as, but not limited to, those commercially available from from BD Biosciences (BD Biosciences, San Jose, CA) using a FACSCanto™ flow cytometer (BD Biosciences). Cells can be counted manually using a disposable C-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed by any method known in the art, including, but not limited to, trypan blue staining.

In some cases, the total TIL population can be cryopreserved immediately using the protocols described below. Alternatively, the total TIL population can be REPed and then cryopreserved as described below. Likewise, in the event that genetically modified TILs are to be used in therapy, populations of total or REP TILs can be genetically modified for appropriate treatment.

2. Cell cultures.

In one embodiment, a method of propagating TILs may include using about 5,000 ml to about 25,000 ml of cell culture medium, from about 5,000 ml to about 10,000 ml of cell culture medium, or from about 5,800 ml to about 8,700 ml of cell culture medium. In one embodiment, no more than one type of culture is used to propagate TILs.

- 80 045580 cell media. Any suitable cell culture medium can be used, for example, AIM-V cell culture medium (L-glutamine, 50 μM streptomycin sulfate and 10 μM gentamicin sulfate) (Invitrogen, Carlsbad, Calif.). In this regard, the patented methods have the advantage of reducing the amount of media, as well as the number of types of media required for TIL propagation. In one embodiment, TIL propagation may involve feeding the cells no more frequently than every third or fourth day. Propagating cells in a gas-permeable container simplifies the procedures required for cell propagation by reducing the frequency of feeding required for cell propagation.

In an embodiment, the cell culture medium in the first and/or second gas-permeable container is unfiltered. The use of unfiltered cell media can simplify the procedures required for cell propagation. In one embodiment, beta-mercaptoethanol (BME) is absent from the cell environment in the first and/or second gas-permeable container.

In one embodiment, the duration of the method includes obtaining a sample of tumor tissue from a mammal; culturing a sample of tumor tissue in a first gas-permeable container containing a cell medium; obtaining TIL from a tumor tissue sample; propagating the TILs in a second gas-permeable container containing cell media using aAPC for a period of about 14 to about 42 days, such as about 28 days.

In one embodiment, TILs are propagated in gas-permeable containers. Gas-permeable containers have been used to propagate TILs using PBMCs using methods, compositions and devices known in the art, including those described in US Patent Application Publication No. 2005/0106717 A1, the disclosure of which is incorporated herein by reference. In one embodiment, TILs are propagated in gas-permeable bags. In one embodiment, TILs are expanded using a cell expansion system that expands TILs in gas-permeable bags, such as the Xuri Cell Expansion System W25 (GE Healthcare). In one embodiment, TILs are propagated using a cell expansion system that propagates TILs in gas-permeable bags, such as the WAVE bioreactor system, also known as the Xuri W5 cell expansion system (GE Healthcare). In one embodiment, the cell propagation system includes a gas-permeable cell bag with a volume selected from the group consisting of about 100 ml, about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml, about 700 ml, about 800 ml, about 900 ml, about 1 l, about 2 l, about 3 l, about 4 l, about 5 l, about 6 l, about 7 l, about 8 l, about 9 l and about 10 l.

In one embodiment, TILs can be propagated in G-Rex flasks (commercially available from Wilson Wolf Manufacturing). Such embodiments allow the cell population to be expanded from about 5x105 cells/ ^cm2 to 10x106 to 30x106 cells/ ^cm2 . In an embodiment, this occurs without replenishment. In one embodiment, this occurs without feeding while the medium is at a height of about 10 cm in the G-Rex flask. In one embodiment, this occurs without feeding, but with the addition of one or more cytokines. In one embodiment, the cytokine can be added as a bolus without the need to mix the cytokine with the medium. Such containers, devices and methods are known in the art and have been used for propagating TILs, including those described in US Patent Application Publication No. US 2014/0377739 A1, International Publication No. WO 2014/210036 A1, US Patent Application Publication No. US 2013/ 0115617 A1, International Publication No. WO 2013/188427 A1, US Patent Application Publication No. US 2011/0136228 A1, US Patent No. US 8809050 B2, International Publication No. WO 2011/072088 A2, US Patent Application Publication No. US 2016/0208216 A1, US Patent Application Publication No. US 2012/0244133 A1, International Publication No. WO 2012/129201 A1, US Patent Application Publication No. US 2013/0102075 A1, US Patent Application Publication No. US 8956860 B2, International Publication No. WO 2013 /173835 A1, publication of US patent application No. US 2015/0175966 A1, the description of which is incorporated herein by reference. Such processes are also described in Jin et al., J. Immunotherapy, 2012, 35:283-292.

Optional genetic modification of TILs.

In some embodiments, TILs are optionally genetically modified to include additional functional properties, including, but not limited to, a high-affinity T cell receptor (TCR), e.g., a TCR targeting a tumor-associated antigen such as MAGE-1, HER2, or NY-ESO-1, or a chimeric antigen receptor (CAR) that binds to a tumor-associated cell surface molecule (eg, mesothelin) or a lineage-specific cell surface molecule (eg, CD 19).

IV. Methods of treating patients.

Treatment methods begin with collecting the original TILs and culturing the TILs. Such methods are described in the art, for example, in Jin et al., J. Immunotherapy, 2012, 35(3):283-292, the entire contents of which are incorporated herein by reference. Options for implementing the treatment methods are described in the sections below, including examples.

Expanded TILs produced by the methods described herein, including, for example

- 81 045580 measures, methods described for steps AF above or in accordance with steps AF (also presented, for example, in Fig. 2A and/or 9), find specific application for the treatment of patients suffering from cancer (for example, as described in Goff, et al., J. Clinical Oncology, 2016, 34(20):2389-239, and in the supplement; the entire content is incorporated herein by reference). In some embodiments, TILs are grown from resected metastatic melanoma as previously described (see Dudley, et al., J Immunother., 2003, 26:332-342; the entire contents are incorporated herein by reference). A fresh tumor can be excised under sterile conditions. A representative sample can be selected for formal pathology analysis. Single fragments with a volume of 2 mm ³ to 3 mm ³ can be used. In some embodiments, 5, 10, 15, 20, 25, or 30 samples are obtained for each patient. In some embodiments, 20, 25, or 30 samples are obtained for each patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained for each patient. In some embodiments, 24 samples are obtained for each patient. Samples can be plated in individual wells of a 24-well plate, maintained in high dose IL-2 growth medium (6000 IU/ml) and monitored for tumor destruction and/or TIL proliferation. Any tumor with viable cells remaining after treatment can be enzyme digested to a single-cell suspension and cryopreserved as described herein.

In some embodiments, successfully growing TILs can be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and tested against autologous tumor, if present. TILs can be considered active if overnight coculture results in the secretion of interferon gamma (IFN-γ) at levels greater than 200 pg/ml and twice the background level. (Goff, et al., JImrmmother., 2010, 33:840-847; the entire contents are incorporated herein by reference). In some embodiments, crops with evidence of autologous activity or suitable growth patterns can be selected for a second propagation (eg, the second propagation shown in step D of Figures 2A and/or 9), including a second propagation that is sometimes referred to as rapid propagation (REP). In some embodiments, expanded TILs with high autologous activity (eg, high rates of proliferation during a second expansion) are selected for additional second expansion. In some embodiments, TILs with high autologous activity (eg, a high degree of proliferation during the second expansion, as shown in step D of FIG. 2A and/or 9) are selected for additional second expansion, as shown in step D of FIG. 2A and/or 9.

In some embodiments, the patient is not immediately subjected to ACT (adoptive cell transfer), e.g., in some embodiments, after tumor acquisition and/or first expansion, the cells are not used immediately. In some embodiments, TILs can be cryopreserved and thawed up to 2 days prior to administration to a patient. In some embodiments, TILs can be cryopreserved and thawed 1 day prior to administration to a patient. In some embodiments, TILs can be cryopreserved and thawed immediately prior to administration to a patient.

Cellular phenotypes of cryopreserved TIL samples from the infusion bag can be analyzed by flow cytometry (eg, FlowJo) for surface markers CD3, CD4, CD8, CCR7 and CD45RA (BD BioSciences), as well as any of the methods described herein. Serum cytokines can be determined by standard enzyme-linked immunosorbent assay methods. An increase in serum IFN-γ levels is defined as a concentration greater than 100 pg/ml and an increase in baseline IFN-γ levels of at least 4 times, or at least 3 times, or at least 2 times, or at least 1 time. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 1000 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 200 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 250 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 300 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 350 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 400 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 450 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 500 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 550 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 600 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 650 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 700 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 750 pg/ml. In some embodiments, the implementation

- 82 045580 The serum level of IFN-γ is determined based on a concentration of more than 800 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 850 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 900 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 950 pg/ml. In some embodiments, an increase in serum IFN-γ level is determined based on a concentration greater than 1000 pg/ml.

In some embodiments, TILs produced by methods provided herein, such as those presented in FIG. 2A and/or 9 are characterized by an unexpected increase in the clinical effectiveness of TILs. In some embodiments, TILs produced by methods provided herein, such as the methods presented in FIG. 2A and/or 9 have increased clinical efficacy compared to TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIGS. 2A and/or 9. In some embodiments, methods other than those described herein include methods referred to as the 1C process and/or the Gen 1 process. In some embodiments, improved clinical efficacy is determined based on DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by methods provided herein, such as the methods presented in FIG. 2A and/or 9 demonstrate a similar time to response and safety profile compared to TILs produced by methods other than those described herein, including, for example, methods other than those presented in FIGS. 2A and/or 9, for example, by the Gen 1 process.

In some embodiments, interferon gamma (IFN-γ) is an indicator of treatment efficacy and/or increased clinical efficacy. In some embodiments, IFN-γ in the blood of TIL-treated subjects is an indicator of the presence of active TILs. In some embodiments, an efficacy assay based on determining IFN-γ production is used. IFN-γ production is another indicator of cytotoxic potential. IFN-γ production can be measured by determining IFN-γ cytokine levels in blood, serum, or TILs ex vivo from a subject treated with TILs produced by the methods of the present invention, including the methods described, for example, in FIG. 2A and/or 9. In some embodiments, an increase in the level of IFN-γ is an indicator of the effectiveness of treatment of a patient treated with TILs produced by the methods of the present invention. In some embodiments, the level of IFN-γ is increased by one-fold, two-fold, three-fold, four-fold, or five-fold, or more compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than from those proposed herein, including, for example, methods other than those presented in FIG. 2A and/or 9. In some embodiments, IFN-γ secretion is increased one-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, , methods other than those shown in FIG. 2A and/or 9. In some embodiments, IFN-γ secretion is increased two-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, , methods other than those shown in FIG. 2A and/or 9. In some embodiments, IFN-γ secretion is increased threefold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, , methods other than those shown in FIG. 2A and/or 9. In some embodiments, IFN-γ secretion is increased fourfold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, , methods other than those shown in FIG. 2A and/or 9. In some embodiments, IFN-γ secretion is increased fivefold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, , methods other than those shown in FIG. 2A and/or 9. In some embodiments, the level of IFN-γ is measured using a Quantikine ELISA kit. In some embodiments, the level of IFN-γ is measured in TILs ex vivo from a subject treated with TILs produced by the methods of the present invention, including the methods described, for example, in FIG. 2A and/or 9. In some embodiments, the level of IFN-γ is measured in the blood of a subject treated with TILs produced by the methods of the present invention, including the methods described, for example, in FIG. 2A and/or 9. In some embodiments, the level of IFN-γ is measured in serum TILs from a subject treated with TILs produced by the methods of the present invention, including the methods described, for example, in FIG. 2A and/or 9.

In some embodiments, TILs produced by the methods of the present invention are

- 83 045580 including the methods described, for example, in FIG. 2A and/or 9 are characterized by increased polyclonality compared to TILs produced by other methods, including methods not shown in FIG. 2A and/or 9, such as, for example, methods called process 1C. In some embodiments, significantly improved polyclonality and/or increased polyclonality is an indicator of treatment efficacy and/or increased clinical efficacy. In some embodiments, polyclonality means diversity in the T cell repertoire. In some embodiments, an increase in polyclonality may be an indicator of the effectiveness of treatment associated with the administration of TILs produced by the methods of the present invention. In some embodiments, the polyclonality is increased by one-fold, two-fold, ten-fold, 100-fold, 500-fold, or 1000-fold compared to TILs produced by methods other than those provided herein, including, for example, methods different from those shown in Fig. 2A and/or 9. In some embodiments, the polyclonality is increased by one-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, methods different from those shown in Fig. 2A and/or 9. In some embodiments, the polyclonality is increased twofold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, methods different from those shown in Fig. 2A and/or 9. In some embodiments, the polyclonality is increased tenfold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, methods different from those shown in Fig. 2A and/or 9. In some embodiments, the polyclonality is increased 100-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, methods different from those shown in Fig. 2A and/or 9. In some embodiments, the polyclonality is increased 500-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, methods different from those shown in Fig. 2A and/or 9. In some embodiments, the polyclonality is increased 1000-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those provided herein, including, for example, methods different from those shown in Fig. 2A and/or 9.

Efficacy measures may include quantitative measures of disease control rate (DCR) as well as overall response rate (ORR), as is known in the art and also described herein.

A. Methods of treating cancer and other diseases.

The compositions and methods described herein can be used to treat diseases. In one embodiment, they are intended for use in the treatment of hyperproliferative diseases, such as cancer, in adult patients or pediatric patients. They may also be used in the treatment of other diseases described in this document and the following sections.

In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the hyperproliferative disease is a solid malignant tumor. In some embodiments, the solid malignant tumor is selected from the group consisting of anal cancer, bladder cancer, breast cancer (including triple negative breast cancer), bone cancer, human papillomavirus (HPV) cancer, central cancer associated nervous system (including ependymoma, medulloblastoma, neuroblastoma, pineoblastoma and primitive neuroectodermal tumor), cervical cancer (including squamous cell cervical cancer, adenosquamous cervical cancer and cervical adenocarcinoma), colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastroesophageal junction cancer, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumor, glioblastoma, glioma, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC), hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, oropharyngeal cancer and pharyngeal cancer), kidney cancer, liver cancer, lung cancer (including non-small cell lung cancer (NSCLC) and small cell lung cancer), melanoma (including uveal melanoma, choroidal melanoma, ciliary body melanoma or iris melanoma), mesothelioma (including malignant pleural mesothelioma), ovarian cancer, pancreatic cancer (including pancreatic ductal adenocarcinoma), penile cancer, rectal cancer, kidney cancer, renal cell carcinoma, sarcoma (including Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma and other bone and soft tissue sarcomas ), thyroid cancer (including anaplastic thyroid cancer), uterine cancer and vaginal cancer.

In some embodiments, the hyperproliferative disease is a hematologic malignancy. In some embodiments, hematologists

- 84 045580 cancer is selected from the group consisting of chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, follicular lymphoma, mantle cell lymphoma and multiple myeloma. In some embodiments, the present invention relates to a method of treating a patient suffering from cancer, wherein the cancer is a hematologic malignancy. In some embodiments, the present invention provides a method of treating a patient suffering from cancer with TILs, MILs, or PBLs modified to express one or more CCRs, wherein the cancer is a hematologic malignancy. In some embodiments, the present invention provides a method of treating a patient suffering from cancer with MIL or PBL modified to express one or more CCRs, wherein the cancer is a hematologic malignancy.

In one embodiment, the cancer is one of the above cancers, including solid cancers and hematologic malignancies, that is relapsed or refractory to treatment with at least one prior therapy, including chemotherapy, radiation therapy, or immunotherapy. In one embodiment, the cancer is one of the above cancers that is recurrent or refractory to treatment with at least two prior therapies, including chemotherapy, radiation therapy and/or immunotherapy. In one embodiment, the cancer is one of the above cancers that is recurrent or refractory to treatment with at least three prior therapies, including chemotherapy, radiation therapy and/or immunotherapy.

In some embodiments, the cancer is a microsatellite instability-high (MSI-H) cancer or a mismatch repair deficiency (dMMR) cancer. Thus, MSI-H and dMMR cancer and its testing are described in Kawakami, et al., Curr. Treat. Options Oncol. 2015, 16, 30, the description of which is incorporated herein by reference.

In some embodiments, the present invention provides a method of treating a patient suffering from cancer with TILs, MILs, or PBLs modified to express one or more CCRs, wherein the patient is a human. In some embodiments, the present invention provides a method of treating a patient suffering from cancer with TILs, MILs, or PBLs modified to express one or more CCRs, wherein the patient is a non-human subject. In some embodiments, the present invention provides a method of treating a patient suffering from cancer with a TIL, MIL, or PBL modified to express one or more CCRs, wherein the patient is a pet. In some embodiments, the present invention provides a method of treating a patient suffering from cancer with TILs, MILs, or PBLs modified to express one or more CCRs, wherein the patient is a primate, horse, dog, or cat.

In some embodiments, the present invention provides a method of treating a patient suffering from cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor and/or a MEK inhibitor. In some embodiments, the present invention provides a method of treating a patient suffering from cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention provides a method of treating a patient suffering from cancer, wherein the cancer is refractory to treatment with a MEK inhibitor selected from the group consisting of trametinib, cobimetinib, bimetinib, selumetinib, pimasertinib, refametinib, and pharmaceutically acceptable salts or solvates thereof. In some embodiments, the present invention provides a method of treating a patient suffering from cancer, wherein the cancer is refractory to treatment with a BRAF inhibitor selected from the group consisting of vemurafenib, dabrafenib, encorafenib, sorafenib and pharmaceutically acceptable salts or solvates thereof, and a MEK inhibitor, selected from the group consisting of trametinib, cobimetinib, binimetinib, selumetinib, pimasertinib, refametinib and pharmaceutically acceptable salts or solvates thereof.

In some embodiments, the present invention relates to a method of treating a patient suffering from cancer, wherein the cancer is childhood cancer.

In some embodiments, the present invention relates to a method of treating a patient suffering from cancer, wherein the cancer is uveal melanoma.

In some embodiments, the present invention provides a method of treating a patient suffering from cancer, wherein the uveal melanoma is choroidal melanoma, ciliary body melanoma, or iris melanoma.

In some embodiments, the present invention relates to a method of treating a patient suffering from cancer, wherein the childhood cancer is neuroblastoma.

In some embodiments, the present invention provides a method of treating a patient suffering from cancer, wherein the childhood cancer is sarcoma.

In some embodiments, the present invention relates to a method of treating a patient suffering from cancer, wherein the sarcoma is osteosarcoma.

In some embodiments, the present invention relates to a method of treating a patient suffering from cancer, wherein the sarcoma is a soft tissue sarcoma.

In some embodiments, the present invention provides a method of treating a patient suffering from cancer, wherein the soft tissue sarcoma is rhabdomyosarcoma, Ewing's sarcoma, or primitive neuroectodermal tumor (PNET).

In some embodiments, the present invention relates to a method of treating a patient suffering from cancer, wherein the childhood cancer is a cancer associated with the central nervous system (CNS). In some embodiments, childhood cancer is refractory to chemotherapy treatment. In some embodiments, childhood cancer is refractory to treatment with radiation therapy. In some embodiments, childhood cancer is refractory to treatment with dinutuximab.

In some embodiments, the present relates to a method of treating a patient suffering from cancer, wherein the CNS-related cancer is a medulloblastoma, pineoblastoma, glioma, ependymoma, or glioblastoma.

The compositions and methods described herein can be used in a method of treating cancer, wherein the cancer is refractory or resistant to treatment with an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient is a patient with primary refractoriness to an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient does not have a previous response to an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the patient experiences a prior response to an anti-PD-1 or anti-PD-L1 antibody, followed by progression of the patient's cancer. In some embodiments, the cancer is refractory to an anti-CTLA4 antibody and/or an anti-PD-1 antibody or an anti-PD-L1 antibody in combination with at least one chemotherapy agent. In some embodiments, the precursor chemotherapeutic agent is carboplatin, paclitaxel, pemetrexed, and/or cisplatin. In some previous embodiments, the chemotherapeutic agent(s) is a platinum doublet chemotherapeutic agent. In some embodiments, the platinum doublet therapy includes a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine, and a taxane (including, for example, paclitaxel, docetaxel, or nab -paclitaxel). In some embodiments, the platinum doublet chemotherapy agent is in combination with pemetrexed.

In some embodiments, the NSCLC is PD-L1 negative and/or comes from a patient suffering from cancer that expresses PD-L1 with a tumor proportion score (TPS) of less than 1%, as described elsewhere herein.

In some embodiments, NSCLC is refractory to a combination therapy comprising an anti-PD-1 or anti-PD-L1 antibody and a platinum doublet therapy, wherein the platinum doublet therapy includes:

i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, ii) and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine and a taxane (including, for example, paclitaxel, docetaxel or nab-paclitaxel).

In some embodiments, NSCLC is refractory to a combination therapy comprising an anti-PD-1 or anti-PD-L1 antibody, pemetrexed, and a platinum doublet therapy, wherein the platinum doublet therapy includes:

i) a first chemotherapeutic agent selected from the group consisting of cisplatin and carboplatin, ii) and a second chemotherapeutic agent selected from the group consisting of vinorelbine, gemcitabine and a taxane (including, for example, paclitaxel, docetaxel or nab-paclitaxel).

In some embodiments, NSCLC is treated with an anti-PD-1 antibody. In some embodiments, NSCLC is treated with an anti-PD-L1 antibody. In some embodiments, the patient with NSCLC has not received treatment. In some embodiments, NSCLC is not treated with an anti-PD-1 antibody. In some embodiments, NSCLC is not treated with an anti-PD-L1 antibody. In some embodiments, NSCLC has been previously treated with a chemotherapy agent. In some embodiments, NSCLC was previously treated with a chemotherapy agent but is no longer treated with a chemotherapy agent. In some embodiments, the NSCLC patient has not received anti-PD-1/PD-L1 antibody treatment. In some embodiments, the NSCLC patient has low PD-L1 expression. In some embodiments, the patient with NSCLC has not received treatment for NSCLC or has been treated following chemotherapy but has not received anti-PD-1/PD-L1 antibody treatment. In some embodiments, the NSCLC patient has received no treatment or has been treated following chemotherapy but has not received anti-PD-1/PD-L1 antibody treatment and has low PD-L1 expression. IN

- 86 045580 In some embodiments, the patient with NSCLC has bulky disease at baseline. In some embodiments, the subject has bulky disease at baseline and has low PD-L1 expression. In some embodiments, the patient suffering from NSCLC lacks detectable expression of PD-L1. In some embodiments, the patient with NSCLC has been previously treatment-naïve or has been treated following chemotherapy but has not received anti-PD-1/PD-L1 antibody treatment and does not have detectable PD-L1 expression. In some embodiments, the patient has bulky disease at baseline and lacks detectable PD-L1 expression. In some embodiments, the patient with NSCLC has not previously been treated for NSCLC or has been treated following chemotherapy (eg, with a post-chemotherapy agent) but has not received anti-PD-1/PD-L1 antibody treatment, has low PD-L1 expression, and/or bulky disease at baseline. In some embodiments, bulky disease is indicated when the maximum diameter of the tumor exceeds 7 cm, measured in either the transverse or coronal plane. In some embodiments, bulky disease is indicated by the presence of swollen lymph nodes with a short-axis diameter of 20 mm or more. In some embodiments, the chemotherapeutic agent includes a standard therapeutic agent for treating NSCLC.

In some embodiments, PD-L1 expression is determined by a tumor proportion score. In some embodiments, the subject suffering from a refractory NSCLC tumor has a tumor proportion score (TPS) of less than 1%. In some embodiments, the subject suffering from a refractory NSCLC tumor has a TPS of at least 1%. In some embodiments, the subject suffering from refractory NSCLC has previously received anti-PD-1 and/or anti-PD-L1 antibody treatment, and the tumor proportion score was determined prior to said anti-PD-1 and/or anti-PD-L1 antibody treatment. In some embodiments, the subject suffering from refractory NSCLC has previously received anti-PD-L1 antibody treatment and the tumor proportion score was determined prior to said anti-PD-L1 antibody treatment.

In some embodiments, TILs produced by the methods of the present invention, including the methods described, for example, in FIG. 1 are characterized by increased polyclonality compared to TILs obtained by other methods, including methods that are not shown in FIG. 1, such as, for example, methods called 1C process. In some embodiments, significantly improved polyclonality and/or increased polyclonality is an indicator of treatment efficacy and/or increased clinical effectiveness of cancer treatment. In some embodiments, polyclonality means diversity in the T cell repertoire. In some embodiments, an increase in polyclonality may be an indicator of the effectiveness of treatment associated with the administration of TILs produced by the methods of the present invention. In some embodiments, the polyclonality is increased by one-fold, two-fold, ten-fold, 100-fold, 500-fold, or 1000-fold compared to TILs produced by methods other than those provided herein, including, for example, methods different from those shown in Fig. 1. In some embodiments, the polyclonality is increased by one-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those proposed herein, including, for example, methods other than those presented in fig. 1. In some embodiments, the polyclonality is increased twofold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those proposed herein, including, for example, methods other than those presented in fig. 1. In some embodiments, the polyclonality is increased tenfold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those proposed herein, including, for example, methods other than those presented in fig. 1. In some embodiments, the polyclonality is increased 100-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those proposed herein, including, for example, methods other than those presented in fig. 1. In some embodiments, the polyclonality is increased 500-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those proposed herein, including, for example, methods other than those presented in fig. 1. In some embodiments, the polyclonality is increased 1000-fold compared to an untreated patient and/or compared to a patient treated with TILs obtained by methods other than those proposed herein, including, for example, methods other than those presented in fig. 1.

In some embodiments, PD-L1 expression is determined by a tumor proportion score using another testing method as described herein. In some embodiments, the subject or patient suffering from an NSCLC tumor has a Tumor Proportion Score (TPS) of less than 1%. In some embodiments, the NSCLC tumor has a TPS of at least 1%. In some embodiments, the subject or patient suffering from NSCLC has previously received treatment with an anti-PD-1 and/or anti-PD-L1 antibody, and the tumor proportion score was determined prior to treatment with the anti-PD-1 and/or anti-PD-L1 antibody. In some embodiments, the subject or patient has previously had NSCLC

- 87 045580 received anti-PD-L1 antibody treatment and the tumor proportion score was determined before anti-PD-L1 antibody treatment. In some embodiments, a subject or patient suffering from a refractory or refractory NSCLC tumor has a tumor share score of less than 1% (TPS). In some embodiments, a subject or patient suffering from a refractory or refractory NSCLC tumor has a TPS of at least 1%. In some embodiments, the subject or patient suffering from refractory or refractory NSCLC has previously received treatment with an anti-PD-1 and/or anti-PD-L1 antibody, and the tumor proportion score was determined prior to treatment with the anti-PD-1 and/or anti-PD-L1 antibody . In some embodiments, the subject or patient suffering from refractory or refractory NSCLC has previously received treatment with an anti-PD-L1 antibody and the tumor proportion score was determined prior to treatment with the anti-PDL1 antibody.

In some embodiments, the NSCLC is an NSCLC that is distinguished by a tumor proportion score (TPS), or the percentage of viable tumor cells in a patient taken prior to anti-PD-1 or anti-PD-L1 antibody therapy, demonstrating partial or complete membrane staining of any intensity, for a protein PD-L1 less than 1% (TPS less than 1%). In one embodiment, the NSCLC is an NSCLC that features a TPS selected from the group consisting of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15% , less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0, 8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09% , less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02% and less than 0.01%. In one embodiment, the NSCLC is an NSCLC that features a TPS selected from the group consisting of about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15% , about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.9%, about 0, 8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, about 0.09% , about 0.08%, about 0.07%, about 0.06%, about 0.05%, about 0.04%, about 0.03%, about 0.02% and about 0.01%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 1%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.9%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.8%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.7%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.6%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.5%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.4%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.3%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.2%. In one embodiment, the NSCLC is an NSCLC that has a TPS of 0% to 0.1%. TPS can be measured by methods known in the art, such as those described in Hirsch, et al., J. Thorac. Oncol. 2017, 12, 208-222 or methods used to determine TPS before treatment with pembrolizumab or other anti-PD-1 or anti-PD-L1 drugs. US Food and Drug Administration-approved TPS measurement methods may also be used. In some embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is found on circulating tumor cells.

In some embodiments, partial membrane staining includes 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more. In some embodiments, complete membrane staining includes about 100% membrane staining.

In some embodiments, testing for PD-L1 may include measuring PD-L1 levels in the patient's serum. In these embodiments, measuring PD-L1 in a patient's serum eliminates the uncertainty of tumor heterogeneity and patient discomfort with serial biopsies.

In some embodiments, increased levels of soluble PD-L1 compared to baseline or reference levels correlate with worse prognosis in NSCLC, see, for example, Okuma, et al., Clinical Lung Cancer, 2018, 19, 410-417; Vecchiarelli, et al., Oncotarget, 2018, 9, 17554-17563. In some embodiments, the PD-L1 is exosomal PD-L1. In some embodiments, PD-L1 is expressed on circulating tumor cells.

In one embodiment, the invention provides a method of treating non-small cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a subject or patient in need thereof, wherein the subject or patient has at least one of the following:

PD-L1 predefined tumor proportion score (TPS) less than 1%;

PD-L1 TPS from 1% to 49% or predefined absence of one or more driver mutations;

wherein the driver mutation is selected from the group consisting of an EGFR mutation, an EGFR insertion, a mut

- 88 045580 EGFR exon 20 mutations, KRAS mutations, BRAF mutations, ALK mutations, c-ROS mutations (ROS1 mutation), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A , PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and/or altered MET signaling, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, FBXW7 mutations, CCND3 mutations and GNA11 mutations, the method comprising:

(a) obtaining a first population of TILs from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(b) adding the first population of TILs to the closed system;

(c) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (b) to step (c) occurs without opening the system;

(d) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third a TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second propagation is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (c) to step (d) occurs without opening the system;

(e) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening;

(g) cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process; and (h) administering to the subject or patient a therapeutically effective dose of the third TIL population from the infusion bag in step (g).

In one embodiment, the invention provides a method of treating non-small cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, the method comprising:

(a) testing the patient's tumor for PD-L1 expression and PD-L1 tumor proportion score (TPS);

(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of an EGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation) , ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, P1K3CA, CDKN2A mutations, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and/or altered MET signaling, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARID1A mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, mutations FBXW7, CCND3 mutations and GNA11 mutations;

(c) determining that the patient has a TPS for PD-L1 of from about 1% to about 49% and determining that the patient also does not have driver mutations;

(d) obtaining a first population of TILs from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(e) adding the first TIL population to the closed system;

(f) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (e) to step (f) occurs without opening the system;

(g) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second propagation is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system

- 89 045580 topics;

(h) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (i) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening;

(j) cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process; and (k) administering to the subject or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In one embodiment, the invention provides a method of treating non-small cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, the method comprising:

(a) testing the patient's tumor for PD-L1 expression and PD-L1 tumor proportion score (TPS);

(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of an EGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation) , ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, MET splicing and/or altered MET signaling, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARIDIA mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations, JAK2 mutations, mutations FBXW7, CCND3 mutations and GNA11 mutations;

(c) determining that the patient has a TPS for PD-L1 of less than about 1% and determining that the patient also does not have driver mutations;

(d) obtaining a first population of TILs from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(e) adding the first TIL population to the closed system;

(f) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (e) to step (f) occurs without opening the system;

(g) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third a TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second propagation is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system;

(h) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (i) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening;

(j) cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process; and (k) administering to the subject or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In one embodiment, the invention provides a method of treating non-small cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, the method comprising:

(a) testing the patient's tumor for PD-L1 expression and PD-L1 tumor proportion score (TPS);

(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of an EGFR mutation, an EGFR insertion mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion mutation, RET or RET mergers;

(c) determining that the patient has a TPS for PD-L1 of from about 1% to about 49% and determining that the patient also does not have driver mutations;

(d) obtaining a first population of TILs from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(e) adding the first TIL population to the closed system;

(f) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to obtain a second population of TILs, wherein the first expansion

- 90 045580 breeding is carried out in a closed container providing a first gas-permeable surface area, with the first propagation being carried out over a period of about 3-14 days to produce a second population

TIL, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (e) to step (f) occurs without opening the system;

(g) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third a TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second propagation is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system;

(h) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (i) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening;

(j) cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process; and (k) administering to the subject or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In one embodiment, the invention provides a method of treating non-small cell lung carcinoma (NSCLC) by administering a population of tumor infiltrating lymphocytes (TILs) to a patient in need thereof, the method comprising:

(a) testing the patient's tumor for PD-L1 expression and PD-L1 tumor proportion score (TPS);

(b) testing the patient for the absence of one or more driver mutations, wherein the driver mutation is selected from the group consisting of an EGFR mutation, an EGFR insertion mutation, a KRAS mutation, a BRAF mutation, an ALK mutation, a c-ROS mutation (ROS1 mutation), a ROS1 fusion mutation, RET or RET mergers;

(c) determining that the patient has a TPS for PD-L1 of less than about 1% and determining that the patient also does not have driver mutations;

(d) obtaining a first population of TILs from a tumor resected from a subject or patient by processing a tumor sample obtained from the subject into a plurality of tumor fragments;

(e) adding the first TIL population to the closed system;

(f) performing a first expansion by culturing a first population of TILs in a cell culture medium containing IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for about 3 -14 days to obtain a second TIL population, wherein the second TIL population is at least 50 times larger than the first TIL population, and wherein the transition from step (e) to step (f) occurs without opening the system;

(g) performing a second expansion by adding additional IL-2, OKT-3, and antigen presenting cells (APCs) to the cell culture medium of the second population of TILs to produce a third population of TILs, wherein the second expansion is performed over a period of about 7-14 days to produce a third a TIL population, wherein the third TIL population is a therapeutic TIL population, wherein the second propagation is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (f) to step (g) occurs without opening the system;

(h) collecting the therapeutic population of TILs obtained in step (d), wherein the transition from step (d) to step (e) occurs without opening the system; and (i) transferring the collected TIL population from step (e) to the infusion bag, wherein the transfer from step (e) to (f) occurs without opening;

(j) cryopreserving the infusion bag containing the collected TIL population in step (f) using a cryopreservation process; and (k) administering to the subject or patient a therapeutically effective dose of the third population of TILs from the infusion bag in step (g).

In another embodiment, the invention provides a method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective dose of a therapeutic population of TILs described herein.

In another embodiment, the invention provides a method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective dose of a TIL composition described herein.

In another embodiment, the invention provides a method of treating a subject suffering from cancer as described herein, modified such that prior to administration of a therapeutically effective dose of a TIL therapeutic population and TIL composition described herein, respectively, the subject is placed on a non-myeloablative lymphodepletion regimen.

In another embodiment, the invention provides the

- 91 045580 a method of treating a subject suffering from cancer, modified such that the non-myeloablative lymphodepletion regimen includes the steps of administering cyclophosphamide at a dose of 60 mg/m ² /day for two days followed by administration of fludarabine at a dose of 25 mg/m ² /day for within five days.

In another embodiment, the invention provides a method of treating a subject suffering from cancer as described herein, modified so as to further comprise the step of treating the subject with a high dose IL-2 regimen, beginning on the day after administration of TIL cells to the subject.

In another embodiment, the invention provides a method of treating a subject suffering from cancer as described herein, modified such that a high-dose IL-2 regimen includes administering 600,000 or 720,000 IU/kg as a 15-minute bolus intravenous infusion every eight hours until portability.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer modified such that the cancer is a solid tumor.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer, modified such that the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, cancer breast, human papillomavirus cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer or renal cell carcinoma.

In another embodiment, the invention provides a method of treating a subject suffering from cancer as described herein, modified such that the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer modified such that the cancer is melanoma.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer modified such that the cancer is HNSCC.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer, modified such that the cancer is cervical cancer.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer modified such that the cancer is NSCLC.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer modified such that the cancer is a glioblastoma (including GBM).

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer, modified such that the cancer is gastrointestinal cancer.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer modified such that the cancer is a hypermutated cancer.

In another embodiment, the invention provides a method described herein for treating a subject suffering from cancer modified such that the cancer is a hypermutated childhood cancer.

In another embodiment, the invention provides a therapeutic TIL population described herein for use in a method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective dose of the therapeutic TIL population.

In another embodiment, the invention provides a TIL composition described herein for use in a method of treating a subject suffering from cancer, comprising administering to the subject a therapeutically effective dose of the TIL composition.

In another embodiment, the invention provides a TIL therapeutic population described herein or a TIL composition described herein modified such that before the subject is administered a therapeutically effective dose of the TIL therapeutic population described herein or a TIL composition described herein, the subject is prescribed a non-myeloablative regimen lymphodepletion.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the non-myeloablative lymphodepletion regimen includes the steps of administering cyclophosphamide at a dose of 60 mg/m ² /day in

- 92 045580 for two days followed by the administration of fludarabine at a dose of 25 mg/m ² /day for five days.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition as described herein, modified to further include the step of treating a patient with a high-dose IL-2 regimen beginning the day after administration of the TIL cells to the patient.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that a high-dose IL-2 regimen includes administration of 600,000 or 720,000 IU/kg as a 15-minute bolus intravenous infusion every eight hours until tolerated. .

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is a solid tumor.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer glands, human papillomavirus cancer, head and neck cancer (including head and neck squamous cell carcinoma (HNSCC)), glioblastoma (including GBM), gastrointestinal cancer, kidney cancer or renal cell carcinoma.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is melanoma, HNSCC, cervical cancer, NSCLC, glioblastoma (including GBM), and gastrointestinal cancer.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is melanoma.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is HNSCC.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is cervical cancer.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is NSCLC.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is glioblastoma.

In another embodiment, the invention provides a therapeutic TIL population or TIL composition described herein modified such that the cancer is a gastrointestinal cancer.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is a hypermutated cancer.

In another embodiment, the invention provides a TIL therapeutic population or TIL composition described herein modified such that the cancer is a hypermutated childhood cancer.

In another embodiment, the invention provides use of a TIL therapeutic population described herein in a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of the TIL therapeutic population.

In another embodiment, the invention provides use of a TIL composition described in any of the preceding paragraphs in a method of treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of the TIL composition.

In another embodiment, the invention provides use of a TIL therapeutic population described herein or a TIL composition described herein in a method of treating cancer in a patient, comprising administering to the patient a non-myeloablative lymphodepletion regimen and then administering to the subject a therapeutically effective dose of the TIL therapeutic population described in either the preceding paragraphs, or a therapeutically effective dose of the TIL composition described herein.

1. Cancer treatment methods based on driver mutations.

As used herein, the phrases driver mutation and/or actionable mutation and/or oncogenic driver mutation refer to mutations that are generally considered to be oncogenic drivers (ie, cancer promoters or cancer inducers). Having one or more of these mutations

- 93 045580 has traditionally been used as a target for targeted therapy. Often, driver mutations are investigated and/or analyzed for treatment with targeted therapeutics, including, for example, tyrosine kinase inhibitors (TKIs). Such driver mutations may, in some embodiments, affect or influence the response to first-line therapeutic treatment. The TIL therapies and compositions described herein are effective for treatment regardless of whether such driver mutations are present or absent in the patient or subject. Such driver mutations can be tested and determined by any method known in the art, including whole exome sequencing, or methods aimed at detecting a specific driver mutation.

In some embodiments, the cancer is a cancer that exhibits the presence or absence of one or more driver mutations. In some embodiments, the cancer exhibits the presence of one or more driver mutations. In some embodiments, the cancer exhibits the absence of one or more driver mutations. In some embodiments, the implementation was analyzed for the absence or presence of one or more driver mutations. In some embodiments, one or more driver mutations are missing. In some embodiments, the cancer treatment is not dependent on the presence or absence of one or more driver mutations. In some embodiments, the cancer exhibits one or more driver mutations selected from the group consisting of an EGFR mutation, an EGFR insertion, an EGFR exon 20 mutation, a KRAS mutation, a BRAF mutation, a BRAF V600E mutation, a BRAF V600K mutation, a BRAF V600 mutation, an ALK mutation, a cROS (ROS1 mutation), ROS1 fusions, RET mutations, RET fusions, ERBB2 mutations, ERBB2 amplifications, BRCA mutations, MAP2K1 mutations, PIK3CA, CDKN2A, PTEN mutations, UMD mutations, NRAS mutations, KRAS mutations, NF1 mutations, MET mutations, splicing MET and/or altered MET signaling, TP53 mutations, CREBBP mutations, KMT2C mutations, KMT2D mutations, ARIDIA mutations, RB1 mutations, ATM mutations, SETD2 mutations, FLT3 mutations, PTPN11 mutations, FGFR1 mutations, EP300 mutations, MYC mutations, EZH2 mutations , JAK2 mutations, FBXW7 mutations, CCND3 mutations, and GNA11 mutations; In some embodiments, the cancer exhibits a PD-L1 TPS of less than 1% and has a predetermined absence of one or more driver mutations.

In some embodiments, the cancer is a cancer that is not indicated for treatment with an EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, a c-Ros inhibitor, a RET inhibitor, an ERBB2 inhibitor, a BRCA inhibitor, a MAP2K1 inhibitor, a PIK3CA inhibitor, a CDKN2A inhibitor, a PTEN inhibitor, an UMD, NRAS inhibitor, KRAS inhibitor, NF1 inhibitor, MET inhibitor, TP53 inhibitor, CREBBP inhibitor, KMT2C inhibitor, KMT2D mutation, ARID1A mutation, RB1 inhibitor, ATM inhibitor, SETD2 inhibitor, FLT3 inhibitor, PTPN11 inhibitor, FGFR1 inhibitor, EP300 inhibitor, MYC inhibitor, EZH2 inhibitor, JAK2 inhibitor, FBXW7 inhibitor, CCND3 inhibitor and GNA11 inhibitor.

In some embodiments, the cancer demonstrates a PD-L1 TPS of less than 1% and is not indicated for treatment with an EGFR inhibitor, a BRAF inhibitor, an ALK inhibitor, a c-Ros inhibitor, a RET inhibitor, an ERBB2 inhibitor, a BRCA inhibitor, a MAP2K1 inhibitor, a PIK3CA inhibitor, a CDKN2A inhibitor, PTEN inhibitor, UMD inhibitor, NRAS inhibitor, KRAS inhibitor, NF1 inhibitor, MET inhibitor, TP53 inhibitor, CREBBP inhibitor, KMT2C inhibitor, KMT2D mutation, ARID1A mutation, RB1 inhibitor, ATM inhibitor, SETD2 inhibitor, FLT3 inhibitor, PTPN11 inhibitor, FGFR1 inhibitor , EP300 inhibitor, MYC inhibitor, EZH2 inhibitor, JAK2 inhibitor, FBXW7 inhibitor, CCND3 inhibitor and GNA11 inhibitor.

In some embodiments, the cancer is NSCLC and the EGFR mutation causes the tumor to transform from NSCLC to small cell lung cancer (SCLC).

In some embodiments, the cancer (or biopsy thereof) demonstrates a high tumor mutational load (high TMB; greater than 10 mut/kb) and/or a high level of microsatellite instability (high MSI). In some embodiments, the cancer (or biopsy thereof) demonstrates a high tumor mutational load (high TMB; greater than 10 mut/kb). In some embodiments, the cancer (or biopsy thereof) exhibits a high level of microsatellite instability (high MSI). Methods and systems for assessing tumor mutational load are known in the art. Examples of disclosure of such methods and systems can be found in US Patent No. 9792403, US Patent Application Publication No. US 2018/0363066 A1, International Patent Application Publication No. WO 2013/070634 A1 and WO 2018/106884 A1, as well as in Metzker publication, Nature Biotechnol. Rev. 2010, 11, 31-46, each of which is incorporated herein by reference.

In some embodiments, the EGFR mutation includes, for example, but is not limited to, T790M, Ex19Del, L858R, exon 20 insertion, delE709-T710insD, I744_K745insKIPVAI, K745_E746insTPVAIK, E709X, E709K, E709A, exon 18 deletion, G719X, G719 A, G719S, L861Q, S768I , L747P, A763_764insFQEA, D770_N771insNPG, A763_764insFQEA, P772_H773insDNP exon 20 insertion, H773_V774insNPH exon 20 insertion, S768I, D770_N771insSVD, V769_D770InsASV, p. K745_E746insIPVAIK, p.K745_E746insTPVAIK, p.I744_K745insKIPVAI, D770_N771insNPG,

- 94 045580

P772_H773insPNP, A763_Y764insFQEA and/or EGFR kinase domain duplication (EGFR-KDD). In some embodiments, the EGFR mutation is selected from the group consisting of T790M, Ex19Del, L858R, exon 20 insertion, delE709-T710insD, I744_K745insKIPVAI, K745_E746insTPVAIK, E709X, E709K, E709A, exon 18 deletion, G719X, G719 A, G719S, L861Q, S768I, L747P, A763_764insFQEA, D770_N771insNPG, A763_764insFQEA, P772_H773insDNP exon 20 inserts, H773_V774insNPH exon 20 inserts, S768I, D770_N771insSVD, V769_D770InsASV, p.K745 _E746insIPVAIK,

p.K745_E746insTPVAIK, p.I744_K745insKIPVAI, D770_N771insNPG, P772_H773insPNP, A763_Y764insFQEA and EGFR kinase domain duplication (EGFR-KDD).

In some embodiments, the EGFR mutation is a double mutation, including, but not limited to, L858R/T790M, Ex19Del/T790M, G719X/L861Q, G719X/S768I (or S768I/G719X), S768I/L858R, L858R/E709A, and/or E746_T751delinsA + T790M. In some embodiments, the EGFR mutation is a double mutation selected from the group consisting of L858R/T790M, Ex19Del/T790M, G719X/L861Q, G719X/S768I (or S768I/G719X), S768I/L858R, L858R/E709A, and E746_T751delins A+T790M . Additional properties and methods regarding EGFR mutations are presented in International Patent Application Publication No. WO 2010/020618 A1, which is incorporated herein by reference.

In some embodiments, the ALK mutation includes, but is not limited to, EML4ALK variant 1 (AB274722.1; BAF73611.1), EML4-ALK variant 2 (AB275889.1; BAF73612.1), EML4ALK variant 3a (AB374361.1; BAG55003.1 ), option 3b EML4-ALK (АВ374362.1; BAG55004.1), option 4 EML4ALK (АВ374363.1; BAG75147.1), option 5а EML4-ALK (АВ374364.1; BAG75148.1), option 5b EML4ALK (АВ374365 .1; BAG75149.1), version 6 EML4-ALK (AV462411.1; VAN57335.1), version 7 EML4ALK (AV462412.1; VAN57336.1), KIF5B-ALK (AV462413.1; VAN57337.1), NPM -ALK, TRMZ-ALK, TFGXL-ALK, TEGL-ALK, TFGS-ALK, A11C-ALK, CLTC-ALK, MSN-ALK, TPM4-ALK, MYH9-ALK, RANBP2-ALK, AL017-ALK and CARS-ALK (See, for example, Pulford et al., (2004) J. Cell. Physiol. 199:330358). In addition, one skilled in the art will appreciate that ALK kinase variants can arise depending on the specific fusion event between the ALK kinase and its fusion partner (e.g., EML4 can be fused to at least exon 2, 6a, 6b, 13, 14 and/or 15, as described, for example, in Horn and Pao, J. Clin. Oncol. 2009, 27, 4247-4253, the description of which is incorporated herein by reference.

Additional examples of ALK mutations are described in US Pat. Nos. 9,018,230 and 9,458,508, the disclosures of which are incorporated herein by reference.

In some embodiments, the ROS1 mutation of the present invention is a ROS1 fusion, wherein a portion of the ROS1 polypeptide that includes the kinase domain of the ROS1 protein (or a polynucleotide encoding it) is fused to all or a portion of another polypeptide (or a polynucleotide encoding it), and wherein the name this second polypeptide or polynucleotide is specified in the fusion. In some embodiments, the ROS1 mutation is determined to be a ROS1 fusion protein (e.g., by IHC) and/or a ROS fusion gene (e.g., by FISH) and/or ROS1 mRNA (e.g., by qRT-PCR), which preferably indicates to a ROS1 fusion protein selected from the group consisting of SLC34A2-ROS1 (SLC34A2 exons 13del2046 and 4 fused to ROS1 exons 32 and 34), CD74-ROS1 (CD74 exon 6 fused to ROS1 exons 32 and 34), EZR-ROS1 (EZR exon 10 fused to ROS1 exon 34), TPM3-ROS1 (TRMZ exon 8 fused to ROS1 exon 35), LRIG3-ROS1 (LRIG3 exon 16 fused to ROS1 exon 35), SDC4-ROS1 (exons 2 and 4 SDC4 fused to exon 32 of ROS1, and exon 4 of SDC4 fused to exon 34 of ROS1), GOPC-ROS1, also known as FIG-ROS1, (exon 8 of GOPC fused to exon 35 of ROS1, and exon 4 of GOPC fused to exon 36 ROS1) and G2032R, also known as ROS1G2032R.

Additional descriptions of ROS1 mutations and ROS fusions have been provided in US Patent Application Publications No. US 2010/0221737 A1, US 2015/0056193 A1 and US 2010/0143918 A1, as well as International Patent Application Publication No. WO 2010/093928 A1, each of which incorporated herein by reference. In some embodiments, the RET mutation is a RET fusion or a point mutation.

In some embodiments, the RET point mutation includes, but is not limited to, H6650, K666E, K666M, S686N, G691S, R694Q, M700L, V706M, V706A, E713K, G736R, G748C, A750P, S765P, P766S, E768Q, E768D, L769 L, R770Q, D771N, N777S, V7781, Q781R, L790F, Y791F, Y791N, V804L, V804M, V804E, E805K, E806C, Y806E, Y806F, Y806S, Y806G, Y806C, E818K, S819I, G823E, Y826 M, R833C, P841L, P841P, E843D, R844W, R844Q, R844L, M848T, 1852M A866W, R873W, A876V, L881V, A883F, A883S, A883T, E884K, R886W, S891A, R8970, D898V, E901K, 5904F, S904C2, K9 07E, K907M, R908K, G911D, R912P, R912Q M918T, M918V, M918L6, A919V, E921K, S922P, S922Y, T930M, F961L, R972G, R982C, M1009V, D1017N, V10416 and M1064T.

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In some embodiments, a RET merger is a merger between the RET and a merger partner selected from the group consisting of

BCR, BCR, CLIP

1, KIFSB, CCDC6, RTS1ex9, NCOA4, TRIM33, ERC1, FGFRIOP, MBD1, RAB61P2, PRKARIA, TRIM24, KTN1, GOLGA5, NOOKZ, KIAA1468, TRIM27, AKAR13, FKBP15, SPECCIL, TBL1XR1, CEP55, CUX1, ACBD5, MY H13, PIBF1, KIAA1217 and MPRIP.

Additional descriptions of RET mutations are provided in US Patent No. 10035789, which is incorporated herein by reference in its entirety.

In some embodiments, the BRAF mutation is a BRAF V600E/K mutation. In other embodiments, the BRAF mutation is a mutation other than V600E/K.

In some embodiments, the BRAF mutation other than V600E/K is a kinase-activated mutation, a kinase-impairing mutation, or a non-kinase mutation, and combinations thereof. In some embodiments, the kinase-activated mutation is selected from the group consisting of R4621, 1463S, G464E, G464R, G464V, G466A, G469A, N58, E586K, F595L, L597Q, L597R, L5975, L597V, A598V, T599E, V600 R, K6O1E , 5602D, A728V and their combinations. In some embodiments, the kinase disrupting mutation is selected from the group consisting of G466E, G466R, G466V, Y472C, K483M, D594A, D594E, D594G, D594H, D594N, D594V, G596R, T599A, 5602A, and combinations thereof. In some embodiments, the non-kinase mutation is selected from the group consisting of T4401, 5467L, G469E, G469R, G4695, G469V, L584F, L588F, V600 K6OldelinsE, 56051, Q609L, E611Q, and combinations thereof. In some embodiments, the BRAF mutation other than V600E/K is selected from the group consisting of a D594 duplication, G469, K6O1E, L597, T599, L485W, F247L, G466V, a BRAF fusion, a BRAF-AGAP3 rearrangement, a BRAF exon 15 cut variant, and their combinations.

In some embodiments, the Met mutation includes a point mutation, deletion mutation, insertion mutation, inversion, aberrant splicing, missense mutation, or gene gain that causes an increase in at least one biological activity of the c-Met protein, tyrosine kinase activity, such as enhanced dimerization receptor homologue, ligand binding formation, body and heterodimer enhancement, etc. The Met mutation can be located in any part of the c-Met genes. In one embodiment, the mutation is in the kinase domain of the c-Met protein, encoded by the c-MET gene. In some embodiments, the c-Met mutations are point mutations at N375, V13, V923, R175, V136, L229, S323, R988, S1058/T1010, and E168.

In some embodiments, the ERBB2 mutation is a point mutation in the amino acid sequence of ERBB2. In some embodiments, the ERBB2 point mutation is a mutation that causes amino acid substitutions, causes mRNA splicing, or is a point mutation in an upstream region. Moreover, said mutation includes a nucleotide mutation causing at least one amino acid substitution selected from the group consisting of Q568E, P601R, I628M, P885S, R143Q, R434Q and E874K.

In some embodiments, the ERBB2 mutation is an amplification of ERBB2. In some embodiments, the ERBB2 amplification includes a point mutation selected from the group consisting of V659E, G309A, G309E, S310F, D769H, D769Y, V777L, P780ms, P780Y781insGSP, V842I, R896C, K753E, and L755S, and can be detected using a polymerase assay chain reaction or other sequencing methods known in the art, such as those described in Bose, et al., Cancer Discov. 2013, 3(2), 224-237; and publications by Zuo, et al. Clin Cancer Res. 2016, 22(19), 4859-4869, the description of which is incorporated herein by reference.

In some embodiments, the BRCA mutation is a mutation in BRCA1 and/or BRCA2, preferably BRCA1, and/or one or more other genes whose protein product is associated with BRCA1 and/or BRCA2 at sites of DNA damage, including ATM, ATR, Chk2, H2AX, 53BP1, NFBD1, Mre11, Rad50, nibrin, BRCA1-associated RING domain (BARD1), Abraxas and MSH2. A mutation in one or more of these genes can result in a gene expression pattern that mimics a mutation in BRCA1 and/or BRCA2.

In some embodiments, the BRCA mutation includes a nonsynonymous mutation. In some embodiments, the BRCA mutation includes a nonsense mutation. In some embodiments, the BRCA mutation includes a frameshift mutation. In some embodiments, the BRCA mutation includes a splicing mutation. In some embodiments, the BRCA mutation is expressed as a mutant mRNA and, ultimately, a mutant protein. In some embodiments, the BRCA1/2 protein is functional. In other embodiments, the BRCA1/2 protein has reduced activity. In other embodiments, the BRCA1/2 protein is non-functional.

As used herein for substitutions, the = sign generally applies to mutations.

- 96 045580 refers to synonymous substitutions, silent codons and/or silent substitutions. Specifically, a synonymous substitution (also called a silent substitution or silent codon) refers to the substitution of one nucleotide base for another in an exon of a protein-coding gene, where the resulting amino acid sequence is not modified. This is because the genetic code is degenerate, meaning some amino acids are coded for by more than one codon of three base pairs. Because some codons for a given amino acid differ by only one base pair from others encoding the same amino acid, a point mutation that replaces the wild-type base with one of the alternatives will result in the same amino acid being included in the polypeptide chain extension during gene translation. In some embodiments, synonymous substitutions and mutations affecting non-coding DNA are often considered silent mutations; however, the mutation does not always occur silently and without any impact. For example, a synonymous mutation can affect transcription, splicing, mRNA transport, and translation, any of which can alter the resulting phenotype, rendering the synonymous mutation non-silent. The substrate specificity of a tRNA for a rare codon can influence translation timing and, in turn, cotranslational protein folding. This is reflected in codon preference, which is observed in many species. A non-synonymous substitution/mutation results in an amino acid change that can be roughly classified as conservative (replacement with an amino acid with similar physicochemical properties), semi-conservative (for example, a negatively charged amino acid to a positively charged one) or radical (a significantly different amino acid from others). In some embodiments, the BRCA mutation is a BRCA1 mutation, which includes, but is not limited to:

P871L, K1183R, D693N, S1634G, E1038G, S1040N, S694= (=: silent codon), M1673I, Q356R, S1436=, L771=, K654Sfs*47, S198N, R496H, R841W, R1347G, H619N, S15 33I, L30=, A622V, Y655Vfs*18, R496C, E597K, R1443*, E23Vfs*17, L30F, EllGfs*3, K339Rfs*2, L512F, D693N, P871S, SI MOG, Q1240*, P1770S, R7=, L52F, T176M, A22 4S, L347=, S561F, E597*, K820E, K893Rfs*107, E962K, M1014I, R1028H, E1258D, E1346K, R1347T, L1439F, H1472R, Q1488*, S1572C, E1602K, R1610C, L1621 =, Q1625*, Q1625=, D1754N, R1772Q, R1856* and any combination thereof.

In some embodiments, the BRCA mutation is a BRCA2 mutation, which includes, but is not limited to:

V2466A, N289H, N991D, S455= (=: silent codon), N372H, H743=, V1269=, S2414=, V2171=, L1521=, T3033Nfs*ll, KI 132=, T3033Lfs*29, R2842C, N1784Tfs*7, K3326*, K3326*, D1420Y, I605Yfs*9, I3412V, A2951T, T3085Nfs*26, R2645Nfs*3, S1013*, T1915M, F3090=, V3244I, A1393V, R2034C, L1356=, E2981Rf s*37, N1784Kfs*3, K3416Nfs *ll, K1691Nfs*15, S1982Rfs*22 and any combination of them.

In some embodiments, the NRAS mutation includes, but is not limited to,

E63K, Q61R, Q61K, G12D, G13D, Q61R, Q61L, Q61K, G12S, G12C, G13R, Q61H, G12V, G12A, Q61L, G13V, Q61H, Q61H, G12R, G13C, Q61P, G13S, G12D, G13A, G13D , A18T, Q61X, G60E, G12S, Q61= (=: silent codon), Q61E, Q61R, A146T, A59T, A59D, Q61=, R68T, A146T, G12A, E62Q, G75=, A91V and any combination thereof.

E132K In some embodiments, the PIK3CA mutation includes replacement mutations, deletion mutations, and insertion mutations. In some embodiments, mutations occur in the helical domain of PIK3CA and its kinase. In other embodiments, in the P85BD domain of PIK3CA. In some embodiments, the PIK3CA mutation occurs in exons 1, 2, 4, 5, 7, 9, 13, 18, and 20. In some embodiments, the PIK3CA mutation occurs in exons 9 and 20. In other embodiments, the PIK3CA mutation is a combination of any mutations listed above. Any combination of these exons can be tested, optionally in combination with testing of other exons. Testing for mutations can be done across the entire coding sequence or can focus on regions where mutations cluster. Specific mutation foci occur at nucleotide positions 1624, 1633, 1636, and 3140 of the PIK3CA coding sequence.

In some embodiments, the size of the PIK3CA mutation is small, ranging from 1 to 3 nucleotides. In some embodiments, PIK3CA mutations include, but are not limited to:

G1624A, G1633A, C1636A, A3140G, G113A, Т1258С, G3129T,

S3139T, E542K, E545K, Q546R, H1047L, H1047R and G2702T.

In some embodiments, the MAP2K1 mutation is a somatic MAP2K1 mutation, optionally a MAP2K1 mutation, that increases MEK1 levels. In some embodiments, the MAP2K1 mutation is a mutation in one or more genes associated with the RAS/MAPK pathway, including: HRAS, KRAS, NRAS, ARAF, BRAF, RAF1, MAP2K2, MAPK1, MAPK3, MAPK3. In some embodiments, the MAP2K1 mutation is in one or more genes selected from the group consisting of RASA, PTEN, ENG, ACVRL1, SMAD4, GDF2, or combinations thereof.

In some embodiments, the MAP2K1 mutation includes, but is not limited to,

P124S, Q56P, K57N, E203K, G237*, P124L, G128D, D67N, K57E, E102_I103del,

C121S, K57T, K57N, Q56P, P124L, K57N, G128V, Q58_E62del, F53L, 1126= I103_K104del and any combination thereof.

In some embodiments, the KRAS mutation includes a nonsynonymous mutation. In some embodiments, the KRAS mutation includes a nonsense mutation. In some embodiments, the KRAS mutation includes a frameshift mutation. In some embodiments, the KRAS mutation includes a splicing mutation. In some embodiments, the KRAS mutation is expressed as a mutant mRNA and, ultimately, a mutant protein. In some embodiments, the mutant KRAS protein is functional. In other embodiments, the mutant KRAS protein has reduced activity. In other embodiments, the mutant KRAS protein is nonfunctional.

In some embodiments, the KRAS mutation includes, but is not limited to,

G12D, G12V, G13D, G12C, G12A, G12S, G12R, G13C, Q61H, A146T, Q61R, Q61H, Q61L,

G13S, A146V, Q61K, G13R, G12F, K117N, G13A, G13V, A59T, V14I, K117N, Q22K, Q61P, A146P, G13D, L19F, L19F, Q61K, G12V, G60=, G12=, G13=, A18D, T58I , Q61E, E63K, G12L, G13V, A59G, G60D, G10R, GlOdup, D57N, A59E, , V14G, D33E, G12I, G13dup and any combination thereof, where = indicates the encoding of silence.

In some embodiments, the NF1 mutation includes replacement mutations, deletion mutations, missense mutations, aberrant splicing mutations, and insertion mutations. In some embodiments, the NF1 mutation is a loss of function (LOF) mutation. In some embodiments, the NF1 mutation is selected from the group consisting of R1947X (C5839T), R304X, exon 37 mutation, exon 4b mutation, exon 7 mutation, exon 10b mutation, and exon 10c mutation (e.g., 1570GDT, E524X).

In some embodiments, the CDKN2A mutation includes, but is not limited to:

R24P, D108G, D108N, D108Y, G125R, P114L, R80*, R58*, H83Y, WHO*, P114L,

E88*, WHO*, E120*, D108Y, D84Y, D84N, E69*, P81L, Q50*, L78Hfs*41, D108N, S12*, P48L, E61*, Y44*, E88K, R80*, D84G, L16Pfs*9 , Y129*, D108H, A148T, A36G, A102V, W15*, H83R, A57V, E33*, D74Y, A76V, E153K, D74N, H83D, V82M, R58*, Y129*, El 19*, Y44*, D74A, T18_A19dup , Y44Lfs*76, L32_L37del, V28_E33del, D14_L16del, A68T or any combination thereof.

In some embodiments, the PTEN mutation includes a nonsynonymous mutation. In some embodiments, the PTEN mutation includes a nonsense mutation. In some embodiments, the PTEN mutation includes a frameshift mutation. In some embodiments, the PTEN mutation includes a splicing mutation. In some embodiments, the mutant PTEN is expressed as mRNA and ultimately protein. In some embodiments, the mutant PTEN protein is functional. In other embodiments, the mutant PTEN protein has reduced activity. In other embodiments, the mutant PTEN protein is non-functional. In some embodiments, the PTEN mutation includes, but is not limited to,

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R130Q, R13OG, T319*,

R233*, R130*, K267Rfs*9, N323Mfs*21, N323Kfs*2, R173C, R173H, R335*, Q171*, Q245*, E7*, D268Gfs*30, R130Q, Q214*, R130L, C136R, Q298*, Q17*, H93R, P248Tfs*5, I33del, R233*, E299*, G132D, Y68H, T319Kfs*24, N329Kfs*14, V166Sfs*14, V290*, T319Nfs*6, R142W, P38S, A126T, H61R, F278L , S229*, R130P, G129R, R13OQfs*4, P246L, R130*, G165R, C136Y, R173C, I101T, Y155C, D92E, K164Rfs*3, N184Efs*6, G129E, R130G, G36R, F341V, H123Y, C124S, M35VG127E, G165E and any combination thereof.

In some embodiments, the TP53 mutation includes, but is not limited to,

R175H, G245S, R248Q, R248W, R249S, R273C, R273H, R282W, C135Y, C141Y, P151S,

V157F, R158L, Y163C, V173L, V173M, C176F, H179R, H179Y, H179Q, Y205C, Y220C,

Y234C, M237I, C238Y, S241F, G245D, G245C, R248L, R249M, V272M, R273L, P278L, R280T, E285K, E286K, R158H, C176Y, I195T, G214R, G245V, G266R, G266 E, P278S, R280K or any combination thereof. In some additional embodiments, the TP53 mutation is selected from the group consisting of: G245S; R249S; R273C; R273H; C141Y, V157F, R158L, Y163C, V173L, V173M, Y205C, Y220C, G245C, R249M, V272M, R273L and E286K. In some embodiments, the TP53 mutation includes one or more of the mutations listed above.

In some embodiments, the CREBBP mutation includes, but is not limited to,

R1446C, R1446H, S1680del, I1084Sfs*15, P1948L, I1084Nfs*3, 7R386*, S893L, R1341*, P1423Lfs*36, P1488L, Y15O3H, R1664C, A1824T, RI 173*, R136 0*, Y1450C, H2228D, S71L, P928 =, D1435N, W1502C, Y1503D, R483*, R601Q, S945L, RI 103*, R1288W, R1392*, C1408Y, D1435G, R1446L, H1485Y, Q1491K, Q96*, L361M, L524Wfs*6, Q540* , Q1073*, A1100V or any combination of them.

In some embodiments, the KMT2C mutation includes, but is not limited to, D348N, P350=, R380L, C391*, P309S, C988F, Y987H, S990G, K2797Rfs*26, V346=, R894Q, R284Q, S806=, R1690=, P986=, A1685S , G315S, Q755*, R909K, T316S, S772L, G838S, L291F, P335=, C988F, Q2680=, E765G, K339N, Y816*, R526P, N729D, G845E, I817Nfs*ll, G892R, C1103*, S3 660L, F4496Lfs* 21, G315C, R886C, D348N, S793=, V919L, R2481S, R2884*, R4549C, M305Dfs*28, T316S, P377=, I455M, T820I, S965=, S730Y, P860S, Q873Hfs*40, R904* ,R2610Q, R4478 * and any combination thereof.

In some embodiments, the KMT2D mutation includes, but is not limited to, L1419P, E640D, E541D, E455D, T2131P, K1420R. P2354Lfs*30, G2493=, Q3612=, 1942= T1195Hfs*17, P4170=, P1194H, G1235Vfs*95, P4563=, P647Hfs*283, L449_P457del, P3557=, Q3603=, R1702*, P64 8Tfs*2, R5501*, R4198*, R4484*, R83Q, R1903*, ,R2685* , R4282*, L5326=, R5432W, R2734*, Q2800*, R2830*, Q3745dup, S4010P, R4904*, G5182Afs*61, R5214H, R1615 *, Q2380* , R2687*, R2771*, V3089Wfs*30, Q3799Gfs*212, R4536*, R5030C, R5048C, R5432Q, A221Lfs*40, A476T, A2119Lfs*25,P2557L, R2801*, Q3913*, R4420 W, G4641=, R5097* and any combination of them.

In some embodiments, the ARIDIA mutation includes, but is not limited to, for example, the subject has an ARID1A mutation selected from the group consisting of C884* (*: nonsense mutation), E966K, Q1411*, F1720fs (fs: frameshift),

G1847fs, C1874fs, D1957E, Q1430, R1721fs, G1255E, G284fs, R1722*, M274fs, G1847fs, P559fs, R1276*, Q2176fs, H203fs, A591fs, Q1322*, S2264*, Q 586*, Q548fs and N756fs.

In some embodiments, the RB1 mutation includes, but is not limited to,

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R320X, R467X, R579X, R455X, R358X, R251X, R787X, R552X, R255X, R556X, Y790X, Q575X, E323X, R661W, R579*, R455*, R556*. R787*, R661W, R445*, R467*, Q217*,Q471*, W195*, Q395*, I680T, E137*, R255*, Q344*, Q62*, E440K, A488V, P777Lfs*33, E322K, R656W, G617Rfs *36, C221*, E440*,Q93*, Q504*, E125*, S834*, E323*, Q685*, S829*, W516*, G435*, Q257*, E79*, S567L, V654M, V654Sfs*14, G100Efs*l 1, K715* and any combination thereof.

In some embodiments, the ATM mutation is a mutation in the ATM gene sequence, including, but not limited to,

10744A>G;10744A>G;

11482G>A; IVS3-558A>T; 146OG; 381delA; IVS8-3delGT; 1028delAAAA; 1120OT;

1930insl6; IVS16+2T>C; 2572T>C; IVS21+1G>A; 3085delA; 3381delTGAC; 3602delTT;

4052delT; 4396OT; 5188OT; 5290delC; 5546delT; 5791G>CCT; 6047A>G; IVS44-1G>T;

6672delGC/6677delTACG; 6736dell l/6749del7; 7159insAGCC; 7671delGTTT; 7705dell4;

7865OT; 7979delTGT; 8177OT; 8545OT; 8565T>A; IVS64+1G>T and 9010del28.

In some embodiments of the present invention, a SETD2 mutation is a change in the sequence of the gene encoding the SETD2 protein when the position of the transcription initiation codon in the mRNA sequence with public access number NCBI NM_014159 is set to 1. In some embodiments, the 7558th G (guanine) is replaced with a T (thymine), 4774th C (cytosine) is replaced by T, 1210th A (adenine) is replaced by T, 4883rd T is replaced by G, 5290th C is replaced by T, 7072nd C is replaced by T, 4144th G replaced by T, 1297th C replaced by T, 755th T replaced by G, 7261st T replaced by G, 6700 replaced by T, 2536th C replaced by T, 7438th C replaced to replace T or there is insertion A at position 3866, insertion T at position 6712, insertion T at position 7572, deletion 913 A, deletion 5619 C, deletion of bases 4603-4604, deletion of base 1, deletion 1936- go C, deletion of bases 3094-3118, insertion of A at position 5289 and deletion of bases 6323-6333.

In some embodiments, the FLT3 mutation includes, but is not limited to, (Q569_E648)ins, D835X, (Q569_E648)delins, (D835J836), D835Y, D835V, D835Y, D835H, T227M, I836del, N676K, D835E, Y597_E598insDYVDFREY, D835E, D835del, F594_D600dup , A680V, D839G, D96=, D835H, V491L, D835E, Q989*, D835V, L561= I836del, P986Afs*27, D7G, D324N, S451F, D835N, L576P, Y597_E598insDVDFREY, V491L, N841 T, D324N, Y572C, R595_L601dup, K663R , N676K, F691L, D835A, I836H, N841K, S993L, L832F, I836M, A66V and any combination thereof.

In some embodiments, the PTPN11 mutation includes, but is not limited to, E76K, A72V, A72T, D61Y, D61V, G60V, E69K, E76G, G507V, S506L, G507A, T73I, E76A, E76Q, S506P, D61N, F71L, E76V, F71L , A72D, V432M, Т472М, P495L, N58Y, F285S, S506A, S189A, А465Т, R502W, G507R, Т511К, D61H, D61G, G507E, G60R, G60A, Q514L, E139D, Y197*, N308D, Q5 14H, Q514H, N58S, E123D, L206=, A465G, P495S, G507R and any combination of them.

In some embodiments, the FGFR1 mutation includes, but is not limited to,

N577K, K687E, N577K, D166del, T371M, R476W, T350= E498K, N577D, D683G, R87C, A154D, N303=, A374V, D550=, S633=, V695L, G728=, R765W, P803S, W19C, P 56=, R113C , V149I, S158L, D166dupR220C, N224Kfs*8, D249N, R281W, R281Q, A299S, S424L, S461F, S467F, R506Q and any combination thereof.

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In some embodiments, the EP300 mutation includes, but is not limited to,

D1399N, Y1414C, M1470Cfs*26, ¥1111*, H2324Pfs*55, R1627W, N2209_Q2213delinsK, Q2268del, L415P, M1470Nfs*3, E1514K, C1201Y, P1452L, S952*, C116 4Y, D1399Y, S507G, Q824*, D1507N, H2324Tfs* 29, P925T, P1440L, W1466C, P1502L, A1629V, R1645*, N1700Tfs*9, P1869L, Q65*, A171V, R202*, R580Q, A627V, Q1082*, N1236Kfs*2, N1286S, R1312*, R1356*, C1385F, H1451L, R1462*, Y1467N, Y1467H, R1478H, R1627Q, R86*, R370H, R397*, R754C, P842S, I997V, E1014* and any combination thereof.

In some embodiments, the MY C mutation includes, but is not limited to,

E61T, E68I, R74Q, R75N, W135E, W136E, V394D, L420P, W96E, V325D, L351P, MYC protein with deletion of 41 amino acids HaN-koitse (dN2MYC), N26S, S16IL, P74L, V7M, F153S, E54D, P246, L16 4V , P74S, A59V, T73I, Р72Т, Т73А, H374R, P17S, T73N, S264N, P72S, Q52del, S21T, Р74А, S107N, P75S, S77P, P261S, P74Q, S190R, А59Т, F153C, Р75Н, T73I, S 77F,N11S , S21N, P78L, P72L, N9K, S190N, S267F, T73P, P78S, G105D, S187C, L71M, Q10H, L191x, Q50x, L191F, R25K, F130L, Y27S, D195N, D2G, V20A, V6G, V20I, D2 H,P75A , G152D, P74T, C40Y, E8K, Q48x and any combination thereof.

In some embodiments, an EZH2 mutation is associated with altered histone methylation patterns. In some embodiments, the EZH2 mutation converts amino acid Y641 (equivalent to Y646, the catalytic domain) to F, N, H, S, or C, resulting in hypertrimethylation of HZK27 and triggering lymphogenesis. In some embodiments, the EZH2 mutation includes mutations in the EZH2 SET domain, overexpression of EZH2, overexpression of other PRC2 subunits, loss-of-function mutations in histone acetyltransferases (HATs), and loss-of-function of MLL2. Cells heterozygous for the EZH2 Y646 mutation result in hypertrimethylation of H3K27 compared with cells homozygous for the wild-type (WT) EZH2 protein or cells homozygous for the Y646 mutation.

In some embodiments, the EZH2 mutation includes, but is not limited to,

Y646F, Y646N, D185H, Y646F, Y646S, Y646H, R690H, Y646X, E745K, Y646C, V626M,

V679M, R690H, R684H, A682G, E249K, G159R, R288Q, N322S, A692V, R690C, D730* (frame shift due to insertion),

S695L, R684C, М667Т, R288*, S644*,

D192N, K55OT, Q653E, D664G, R347Q, Y646C, G660R, R213C, A255T, S538L, N693K, 155M, R561H, A692V, K515R, Y733*, R63*, Q570*, Q328*, R25Q, T467P A6 56V, T573I, C571Y , E725K, R16W, P577L, F145S, V680M, G686D, G135R, K634E, S652F, R298C, G648E, R566H, L149R, R502Q, Y731D, R313W, N675K, S652C, T374Hfs*3, N152If s*15, E401Kfs*22, K406Mfs *17, E246*, S624C, I146T, V626M, L674S, H694R, A581S and any combination thereof.

In some embodiments, the JAK2 mutation is a mutation in the JAK2 gene, including, but not limited to, the T1923C mutation in combination with the G1920T mutation, the G1920T/C1922T mutation, or the G1920A mutation. In some embodiments, the JAK2 mutation is a mutant JAK2 protein containing one or more substitutions, including, but not limited to, V617F, V617I, R683G, N542_E543del, E543_D544del, R683S, R683X, F537_K539delinsL (in-frame deletion), K539L, N1108S, R11 13H , R1063H, R487C, I540Mfs*3 (deletion-frameshift), R867Q, K539L, G571S, R1113C, R938Q, R228Q, L830*, E1080*, K539L, C618R, R564Q, D1036H, L1088S, H538Nf s*4, D873N, V392M, I682F, L393V, M535I, C618R, T875N, L611V, D319N, L611S, G921S, H538Y, S1035L and any combination thereof.

In some embodiments, the FBXW7 mutation is a point mutation selected from the group consisting of W244* (*: stop codon), R222*, R278*, E192A, S282*, E113D, R465H/C, 726+1 G> splice A, R505C, R479Q, R465C, R367*, R499Vfs*25 (fs*: frame shift), R658*, D600Y, D520N, D520Y and any combination thereof. In additional embodiments, the FBXW7 mutation is a double or triple mutation, including, but not limited to, R479Q and S582L, R465H and S582L, D520N, D520Y and R14Q, and R367* and S582L.

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In some embodiments, the CCND3 mutation includes, but is not limited to, S259A,

R271Pfs*53 (frame shift due to insertion),

E51*, Q260*, P199S, T283A,

Т283Р, V287D, D286_T288del, R271Gfs*33, Q276*, R241Q, D238G, R33P, I290K, I290T, I290R, P267fs, P284S, P284L, P1OOS, E253D, S262I, R14W, R114L, D2 38N, A266E, R167W and any combination thereof .

In some embodiments, the GNA11 mutation includes, but is not limited to,

Q209L, R183C, Т257=, R183C, G208Afs*16, Q209H, R183C, Q209P, Q209R, Q209H, ?Т96=, R210W, R256Q, Т334=, G48D, S53G, Q209P, R213Q and any combination thereof. In some embodiments, the GNA11 mutation has two mutations in exon 4, such as a mutation in VI82 and a mutation in T175, or one or more mutations in exon 5.

2. Combinations with PD-1 and PD-L1 inhibitors.

In some embodiments, TIL therapy provided to patients suffering from cancer may include treatment with therapeutic populations of TILs alone or may include a combination treatment comprising TILs and one or more PD-1 and/or PD-L1 inhibitors.

Programmed death 1 (PD-1) is a 288 amino acid transmembrane immune checkpoint receptor protein expressed by T cells, B cells, natural killer (NK) T cells, activated monocytes, and dendritic cells. PD-1, also known as CD279, belongs to the CD28 family and in humans is encoded by the Pdcd1 gene on chromosome 2. PD-1 consists of one immunoglobulin (Ig) superfamily domain, a transmembrane region, and an intracellular domain containing a tyrosine-based immunoreceptor inhibitory motif ( ITIM) and immunoreceptor tyrosine-based switching motif (ITSM). PD-1 and its ligands (PD-L1 and PD-L2) are known to play a key role in immune tolerance, as described in Keir, et al., Annu. Rev. Immunol. 2008, 26, 677-704. PD-1 provides inhibitory signals that negatively regulate T cell immune responses. PD-L1 (also known as B7-H1 or CD274) and PDL2 (also known as B7-DC or CD273) are expressed on tumor and stromal cells, which can be encountered by activated T cells expressing PD-1, resulting in immunosuppression T cells. PD-L1 is a 290 amino acid transmembrane protein encoded by the Cd274 gene on human chromosome 9. Blocking the interaction between PD-1 and its ligands PD-L1 and PD-L2 using a PD-1 inhibitor, a PD-L1 inhibitor and/or a PD-L2 inhibitor can overcome immune resistance, as shown in recent clinical studies such as those described in publications Topalian, et al., N. Eng. J. Med. 2012, 366, 2443-54. PD-L1 is expressed on many tumor cell lines, while PD-L2 is expressed primarily on dendritic cells and a few tumor lines. In addition to T cells (which inducibly express PD1 upon activation), PD-1 is also expressed on B cells, natural killer cells, macrophages, activated monocytes, and dendritic cells.

In one embodiment, the PD-1 inhibitor may be any PD1 inhibitor or PD-1 blocker known in the art. Specifically, it is one of the PD-1 inhibitors or blockers described in more detail in the following paragraphs. The terms inhibitor, antagonist and blocker are used interchangeably herein in relation to PD-1 inhibitors. For the avoidance of doubt, references herein to a PD-1 inhibitor that is an antibody may refer to the compound or antigen binding fragments, variants, conjugates or biosimilars thereof. For the avoidance of doubt, references herein to a PD-1 inhibitor may also refer to a small molecule compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

In a preferred embodiment, the PD-1 inhibitor is an antibody (ie, an anti-PD-1 antibody), a fragment thereof, including Fab fragments, or a single chain variable fragment (scFv) thereof. In some embodiments, the PD-1 inhibitor is a polyclonal antibody. In a preferred embodiment, the PD-1 inhibitor is a monoclonal antibody. In some embodiments, the PD-1 inhibitor competes for binding to PD-1 and/or binds to an epitope on PD-1. In one embodiment, the antibody competes for binding to PD-1 and/or binds to an epitope on PD-1.

In some embodiments, the PD-1 inhibitor is an inhibitor that binds human PD-1 with a KD of about 100 pM or less, binds human PD-1 with a KD of about 90 pM or less, binds human PD-1 with a KD of about 80 pM, or less, binds human PD-1 with a KD of about 70 pM or less, binds human PD-1 with a KD of about 60 pM or less, binds human PD-1 with a KD of about 50 pM or less, binds human PD-1 with a KD of about 40 pM or less, binds human PD-1 with a KD of about 30 pM or less, binds human PD-1 with a KD of about 20 pM or less, binds human PD-1 with a KD of about 10 pM or less, or binds human PD-1 to KD is about 1 pM or less.

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In some embodiments, the PD-1 inhibitor is an inhibitor that binds to human PD-1 with a kassoc of about 7.5 x 105 l/ms or faster, binds to human PD-1 with a kassoc of about 7.5 x 105 l/ms or faster, binds to human PD-1 with kassoc about 8x105 l/ms or faster, binds to human PD-1 with kassoc about 8.5x105 l/ms or faster, binds to human PD-1 with kassoc about 9x105 l/ms or faster, binds with human PD-1 with kassoc of about 9.5x105 l/ms or faster or binds to human PD-1 with kassoc of about 1x10 ⁶ l/ms or faster.

In some embodiments, the PD-1 inhibitor is an inhibitor that binds to human PD-1 with a kdissoc of about 2 x 10 ^-5 hpi or slower, binds to human PD-1 with a kdissoc of about 2.1 x 10 -5 l/s, or slower, binds to human PD-1 with kdissoc about 2.2x10-5 hpi or slower, binds to human PD-1 with kdissoc about 2.3x10-5 l/s or slower, binds to human PD-1 with kdissoc about 2.4x10-5 l/s or slower, binds to human PD-1 with kdissoc about 2.5x10-5 l/s or slower, binds to human PD-1 with kdissoc about 2.6x10-5 l/s or slower or binds to human PD-1 with kdissoc about 2.7x10-5 l/s or slower, binds to human PD-1 with kdissoc about 2.8x10-5 l/s or slower, binds to human PD-1 with kdissoc about 2.9x10-5 l/s or slower or binds to human PD-1 with kdissoc about 3x10-5 l/s or slower.

In some embodiments, the PD-1 inhibitor is an inhibitor that blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC ₅₀ of about 10 nM or less, blocks or inhibits the binding of human PD-L1 or PD Human -L2 to human PD-1 with an IC ₅₀ of about 9 nM or less, blocks or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC ₅₀ of about 8 nM or less, blocks or inhibits the binding of PD- Human L1 or PD-L2 with human PD-1 with an IC ₅₀ of about 7 nM or less, blocks or inhibits the binding of human PDL1 or PD-L2 to human PD-1 with an IC ₅₀ of about 6 nM or less, blocks or inhibits the binding of PD- Human L1 or PD-L2 with human PD-1 with an IC ₅₀ of about 5 nM or less, blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 4 nM or less, blocks or inhibits binding Human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 3 nM or less, blocks or inhibits or blocks the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC ₅₀ of about 2 nM or less or inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC ₅₀ of about 1 nM or less.

In one embodiment, the PD-1 inhibitor is nivolumab (commercially available as OPDIVO from Bristol-Myers Squibb Co.) or biosimilars, antigen binding fragments, conjugates or variants thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-1 receptor. In one embodiment, the anti-PD-1 antibody is kappaimmunoglobulin G4, an anti-human CD274 antibody. Nivolumab has a Chemical Abstracts Service (CAS) registration number of 946414-94-4 and is also known as 5C4, BMS-936558, MDX-1106 and ONO4538. The preparation and properties of nivolumab are described in US Patent No. 8008449 and International Patent Publication No. WO 2006/121168, the disclosure of which is incorporated herein by reference. The clinical safety and efficacy of nivolumab in various forms of cancer are described in Wang, et al., Cancer Immunol. Res. 2014, 2, 846-56; Page, et al., Ann. Rev. Med., 2014, 65, 185-202; and Weber, et al., J. Clin. Oncology, 2013, 31, 4311-4318, the description of which is incorporated herein by reference. The amino acid sequences of nivolumab are presented in table. 18. Nivolumab has disulfide bonds within the heavy chain at positions 22-96, 140-196, 254-314, 360-418, 22-96, 140-196, 254-314 and 360-418; disulfide bonds within the light chain at positions 23'-88', 134'-194', 23'-88' and 134'-194'; disulfide bonds between the heavy and light chains at positions 127214', 127-214', disulfide bonds between the heavy chain at positions 219-219 and 222-222; and N-glycosylation sites (H CH2 84.4) at positions 290, 290.

In one embodiment, the PD-1 inhibitor comprises a heavy chain represented by SEQ ID NO: 158 and a light chain represented by SEQ ID NO: 159. In one embodiment, the PD-1 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 158 and SEQ ID NO: 159, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In one embodiment, the PD-1 inhibitor contains a heavy and a light chain, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 158 and SEQ ID NO: 159, respectively. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 463 and SEQ ID NO: 159, respectively.

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In one embodiment, the PD-1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of nivolumab. In one embodiment, the heavy chain variable region (VH) of the PD-1 inhibitor contains the sequence shown in SEQ ID NO: 160, and the light chain variable region ( _VL ) of the PD-1 inhibitor contains the sequence shown in SEQ ID NO: 161. or their conservative amino acid substitutions. In one embodiment, the PD-1 inhibitor contains VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In one embodiment, the PD-1 inhibitor contains VH and _VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In one embodiment, the PD-1 inhibitor contains VH and _VL regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In one embodiment, the PD-1 inhibitor contains VH and VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 160 and SEQ ID NO: 161, respectively. In one embodiment, the PD-1 inhibitor contains VH and VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 160 and SEQ ID NO: 161, respectively.

In one embodiment, the PD-1 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 162, SEQ ID NO: 163 and SEQ ID NO: 164, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 165, SEQ ID NO: 166 and SEQ ID NO: 167, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on PD-1 as any of the above antibodies.

In one embodiment, the PD-1 inhibitor is a biosimilar anti-PD-1 monoclonal antibody approved by drug regulatory authorities for nivolumab. In one embodiment, the biosimilar comprises an anti-PD-1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is nivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-1 antibody authorized or submitted for registration, wherein the anti-PD-1 antibody is provided in a formulation that is different from the formulations of the reference drug or reference biological product, wherein the reference drug or reference biological product is is nivolumab. An anti-PD-1 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is nivolumab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is nivolumab.

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Table 18

Amino acid sequences for PD-1 inhibitors related to nivolumab

Identifier Sequence (single letter amino acid symbols) SEQ Ш NO:463 heavy chain nivolumab QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAVIWYDGSKRYY 60 ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS 120 VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS 180

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VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKP 240 KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLT 300 VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC 360 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV 420 MHEALHNHYT QKSLSLSLGK 440 SEQ ID NO: 159 light chain nivolumab EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ GO NO: 160 variable heavy chain nivolumab QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAVIWYDGSKRYY 60 ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSS 113 SEQIDNO:161 variable light chain nivolumab EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIK 107 SEQIDNO:162 Nivolumab heavy chain CDR1 NSGMH 5 SEQTDNO163 Nivolumab heavy chain CDR2 VTWYDGSKRY YADSVKG 17 SEQIDNO:164 Nivolumab heavy chain CDR3 NDDY 4

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SEQIDNO:165 CDR1 light nivolumab chains RASQSVSSYL A 11 SEQIDNO:166 CDR2 easy nivolumab chains DASNRAT 7 SEQIDNO:167 CDR3 easy nivolumab chains QQSSNWPRT 9

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, wherein nivolumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, wherein nivolumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2 .5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg or about 10 mg/kg. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, administration of nivolumab begins 1.2 or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, wherein nivolumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, wherein nivolumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg or about 500 mg. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof, wherein nivolumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat unresectable or metastatic melanoma. In some embodiments, nivolumab is administered for the treatment of unresectable or metastatic melanoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of unresectable or metastatic melanoma at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab is administered for the treatment of unresectable or metastatic melanoma at a dose of about 1 mg/kg followed by ipilimumab at a dose of 3 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks or 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered for the adjuvant treatment of melanoma. In some embodiments, nivolumab is administered for the adjuvant treatment of melanoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the adjuvant treatment of melanoma at a dose of about 480 mg every 4 weeks. In some embodiments, enter

- 107 045580 Nivolumab administration begins 1, 2, 3, 4 or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat metastatic non-small cell lung cancer. In some embodiments, nivolumab is administered for the treatment of metastatic non-small cell lung cancer at a dose of about 3 mg/kg every 2 weeks along with ipilimumab at a dose of about 1 mg/kg every 6 weeks. In some embodiments, nivolumab is administered for the treatment of metastatic non-small cell lung cancer at a dose of about 360 mg every 3 weeks with ipilimumab at a dose of 1 mg/kg every 6 weeks and 2 cycles of platinum doublet chemotherapy. In some embodiments, nivolumab is administered for the treatment of metastatic non-small cell lung cancer at a dose of about 240 mg every 2 weeks or 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat small cell lung cancer. In some embodiments, nivolumab is administered for the treatment of small cell lung cancer at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered for the treatment of malignant pleural mesothelioma at a dose of about 360 mg every 3 weeks with ipilimumab at a dose of 1 mg/kg every 6 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat advanced renal cell cancer. In some embodiments, nivolumab is administered for the treatment of advanced renal cell carcinoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of advanced renal cell carcinoma at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab is administered for the treatment of advanced renal cell carcinoma at a dose of about 3 mg/kg followed by ipilimumab at a dose of about 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks . In some embodiments, nivolumab is administered for the treatment of advanced renal cell carcinoma at a dose of about 3 mg/kg, followed by ipilimumab at a dose of about 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks, 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat classical Hodgkin lymphoma. In some embodiments, nivolumab is administered for the treatment of classical Hodgkin lymphoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of classical Hodgkin lymphoma at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

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In some embodiments, nivolumab is administered to treat recurrent or metastatic squamous cell carcinoma of the head and neck. In some embodiments, nivolumab is administered for the treatment of recurrent or metastatic head and neck squamous cell carcinoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of recurrent or metastatic head and neck squamous cell carcinoma at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered for the treatment of locally advanced or metastatic urothelial carcinoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of locally advanced or metastatic urothelial carcinoma at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR). In some embodiments, nivolumab is administered to treat metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) in adult and pediatric patients. In some embodiments, nivolumab is administered for the treatment of metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) in adult and pediatric patients weighing at least 40 kg at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) in adult and pediatric patients weighing at least 40 kg at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) in pediatric patients weighing less than 40 kg at a dose of about 3 mg/kg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) in adult and pediatric patients weighing at least 40 kg at a dose of about 3 mg/kg followed by ipilimumab at a dose of 1 mg/kg on the same day every 3 weeks for 4 doses, then 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) in adult and pediatric patients weighing at least 40 kg at a dose of about 3 mg/kg followed by ipilimumab at a dose of 1 mg/kg on the same day every 3 weeks for 4 doses, then 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat hepatocellular carcinoma. In some embodiments, nivolumab is administered for the treatment of hepatocellular carcinoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of hepatocellular carcinoma at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab is administered for the treatment of hepatocellular carcinoma at a dose of about 1 mg/kg followed by ipilimumab at a dose of 3 mg/kg on the same day every 3 weeks for 4 doses, then

- 109 045580 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of hepatocellular carcinoma at a dose of about 1 mg/kg followed by ipilimumab at a dose of 3 mg/kg on the same day every 3 weeks for 4 doses, then 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, nivolumab is administered to treat squamous cell carcinoma of the esophagus. In some embodiments, nivolumab is administered for the treatment of esophageal squamous cell carcinoma at a dose of about 240 mg every 2 weeks. In some embodiments, nivolumab is administered for the treatment of esophageal squamous cell carcinoma at a dose of about 480 mg every 4 weeks. In some embodiments, nivolumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, nivolumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, nivolumab can also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, nivolumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In another embodiment, the PD-1 inhibitor includes pembrolizumab (commercially available as KEYTRUDA from Merck & Co., Inc., Kenilworth, NJ, USA) or antigen binding fragments, conjugates or variants thereof. Pembrolizumab has CAS registration number 1374853-91-4 and is also known as lambrolizumab, MK-3475 and SCH-900475. Pembrolizumab has immunoglobulin G4, anti-(human PDCD1 protein (programmed cell death 1)) (human Mus musculus monoclonal heavy chain), disulfide with human Mus musculus monoclonal light chain, dimeric structure. The structure of pembrolizumab can also be described as immunoglobulin G4, anti-(human programmed cell death 1); humanized mouse monoclonal [228-L-Proline(H10-S>P)]γ4 heavy chain (134-218')-disulfide with humanized mouse monoclonal dimer to light chain (226-226:229-229)-bisdisulfide. The properties, use and production of pembrolizumab are described in international patent publication No. WO 2008/156712 A1, US patent No. 8354509 and publications of US patent applications No. US 2010/0266617 A1, US 2013/0108651 A1 and US 2013/0109843 A2, the description of which is included in this document by reference. The clinical safety and efficacy of pembrolizumab in various forms of cancer are described in Fuerst, Oncology Times, 2014, 36, 35-36; Robert, et al., Lancet, 2014, 384, 1109-17; and Thomas, et al., Exp. Opin. Biol. Ther., 2014, 14, 1061-1064. The amino acid sequences of pembrolizumab are presented in table. 19. Pembrolizumab includes the following disulfide bridges: 22-96, 22-96, 23'-92', 23'-92', 134-218', 134-218', 138'-198', 138'-198', 147-203, 147-203, 226-226, 229-229, 261-321, 26G-32G, 367-425 and 367-425, as well as the following glycosylation sites (N): Asn-297 and Asn-297. Pembrolizumab is an IgG4/kappa isotype with a stabilizing mutation S228P in the Fc region; insertion of this mutation into the hinge region of IgG4 prevents the formation of half molecules typically observed for IgG4 antibodies. Pembrolizumab is heterogeneously glycosylated at Asn297 in the Fc domain of each heavy chain, giving an intact antibody molecular weight of approximately 149 kDa. The dominant glycoform of pembrolizumab is the fucosylated form of the agalactodiantennary glycan (G0F).

In one embodiment, the PD-1 inhibitor comprises a heavy chain represented by SEQ ID NO: 168 and a light chain represented by SEQ ID NO: 169. In one embodiment, the PD-1 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively. In one embodiment, the PD-1 inhibitor comprises a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 168 and SEQ ID NO: 169, respectively.

In one embodiment, the PD-1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of pembrolizumab. In one embodiment, the variable region

- 110 045580 the yellow chain (VH) of the PD-1 inhibitor contains the sequence shown in SEQ ID NO: 170, and the variable region of the light chain (V _L ) of the PD-1 inhibitor contains the sequence shown in SEQ ID NO: 171, or conserved thereof amino acid substitutions. In one embodiment, the PD-1 inhibitor contains VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In one embodiment, the PD-1 inhibitor contains VH and _VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In one embodiment, the PD-1 inhibitor contains V _H and V _L regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In one embodiment, the PD-1 inhibitor contains VH and VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively. In one embodiment, the PD-1 inhibitor contains VH and VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively.

In one embodiment, the PD-1 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 172, SEQ ID NO: 173 and SEQ ID NO: 174, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 175, SEQ ID NO: 176 and SEQ ID NO: 177, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on PD-1 as any of the above antibodies.

In one embodiment, the PD-1 inhibitor is a biosimilar anti-PD-1 monoclonal antibody approved by drug regulatory authorities for pembrolizumab. In one embodiment, the biosimilar comprises an anti-PD-1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is pembrolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-1 antibody authorized or submitted for registration, wherein the anti-PD-1 antibody is provided in a formulation that is different from the formulations of the reference drug or reference biological product, wherein the reference drug or reference biological product is is pembrolizumab. An anti-PD-1 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is pembrolizumab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is pembrolizumab.

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Table 19

Amino acid sequences for PD-1 inhibitors related to pembrolizumab

Identifier Sequence (single letter amino acid symbols) SEQIDNO:168 heavy chain pembrolizumab QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGGINPSNGGTNF 60 NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS 120 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS 180 GLYSLSSVVT VPSSSLGTKT YTCNVDHKPS NTKVDKRVES KYGPPCPPCP APEFLGGPSV 240 FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY 300 RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK 360 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL TVDKSRWQEG 420 NVFSCSVMHE ALHNHYTQKS LSLSLGK 447 SEQIDNO:169 light chain EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES 60

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pembrolizumab GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF 120 IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 180 STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 218 SEQIDNO:170 variable heavy chain pembrolizumab QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGGINPSNGGTNF 60 NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS 120 SEQIDNO:171 pembrolizumab variable light chain EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES 60 GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI Kill SEQIDNO:172 Pembrolizumab heavy chain CDR1 NYYMY 5 SEQIDNO:173 Pembrolizumab heavy chain CDR2 GINPSNGGTN FNEKFK 16 SEQIDNO:174 Pembrolizumab heavy chain CDR3 RDYRFDMGFD Y 11 SEQIDNO:175 CDR1 of pembrolizumab light chain RASKGVSTSG YSYLH 15 SEQIDNO:176 CDR2 of pembrolizumab light chain LASYLES 7 SEQIDNO:177 CDR3 of pembrolizumab light chain QHSRDLPLT 9

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2 .5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg or about 10 mg/kg. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, where pembrolizumab is administered at a dose of about 200 mg to about 500 mg. In some variants

- 113 045580 the implementation of the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein nivolumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg or about 500 mg. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof, wherein pembrolizumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered to treat melanoma. In some embodiments, pembrolizumab is administered for the treatment of melanoma at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of melanoma at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered to treat NSCLC. In some embodiments, pembrolizumab is administered for the treatment of NSCLC at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of NSCLC at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered to treat small cell lung cancer (SCLC). In some embodiments, pembrolizumab is administered for the treatment of SCLC at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of SCLC at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered to treat head and neck squamous cell carcinoma (HNSCC). In some embodiments, pembrolizumab is administered for the treatment of HNSCC at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of HNSCC at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of classical Hodgkin lymphoma (cHL) or primary mediastinal large B cell lymphoma (PMBCL) at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of classical Hodgkin lymphoma (cHL) or primary mediastinal large B cell lymphoma (PMBCL) at a dose of about 400 mg every 6 weeks for adults. In some embodiments

- 114 045580 pembrolizumab is administered for the treatment of classical Hodgkin lymphoma (cHL) or primary mediastinal large B-cell lymphoma (PMBCL) at a dose of approximately 2 mg/kg (up to 200 mg) every 3 weeks in pediatrics. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of urothelial carcinoma at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of urothelial carcinoma at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered to treat cancers with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of MSI-H or dMMR cancer at a dose of about 400 mg every 6 weeks for adults. In some embodiments, pembrolizumab is administered for the treatment of MSI-H or dMMR cancer at a dose of about 2 mg/kg (up to 200 mg) every 3 weeks in pediatrics. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) colorectal cancer (CRC) at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of MSI-H or dMMR CRC at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of gastric cancer at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of gastric cancer at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of esophageal cancer at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of esophageal cancer at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

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In some embodiments, pembrolizumab is administered for the treatment of cervical cancer at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of cervical cancer at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of hepatocellular carcinoma (HCC) at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered to treat HCC at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of Merkel cell carcinoma (MCC) at a dose of about 200 mg every 3 weeks for adults. In some embodiments, pembrolizumab is administered for the treatment of MSS at a dose of about 400 mg every 6 weeks for adults. In some embodiments, pembrolizumab is administered for the treatment of MSS at a dose of about 2 mg/kg (up to 200 mg) every 3 weeks in pediatric patients. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of renal cell carcinoma (RCC) at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of RCC at a dose of about 400 mg every 6 weeks with axitinib at a dose of 5 mg orally twice daily. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of endometrial carcinoma at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of endometrial carcinoma at a dose of about 400 mg every 6 weeks with lenvatinib at a dose of 20 mg orally once daily for tumors other than MSI-H or dMMR. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of cancers with high tumor mutational burden (TMB-H) at a dose of about 200 mg every 3 weeks for adults. In some embodiments, pembrolizumab is administered for the treatment of TMB-N cancer at a dose of about 400 mg every 6 weeks for adults. In some embodiments, pembrolizumab is administered for the treatment of TMB-N cancer at a dose of about 2 mg/kg (up to 200 mg) every 3 weeks in pediatrics. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab may also be administered 1, 2, or 3 weeks prior to resection (ie, prior to obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of cutaneous squamous cell carcinoma (cSCC) at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered for the treatment of cSCC at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, pembrolizumab is administered for the treatment of triple negative breast cancer (TNBC) at a dose of about 200 mg every 3 weeks. In some embodiments, pembrolizumab is administered to treat TNBC at a dose of about 400 mg every 6 weeks. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In one embodiment, if the patient or subject is an adult, i.e., treatment for an adult indication, an additional dosage regimen of 400 mg every 6 weeks may be administered. In some embodiments, pembrolizumab administration begins 1, 2, 3, 4, or 5 days after IL-2 administration. In some embodiments, pembrolizumab administration begins 1, 2, or 3 days after IL-2 administration. In some embodiments, pembrolizumab may also be administered 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, pembrolizumab can also be administered 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In one embodiment, the PD-1 inhibitor is a commercially available anti-PD-1 monoclonal antibody, such as the anti-m-PD-1 clones J43 (Cat. No. BE0033-2) and RMP1-14 (Cat. No. BE0146) (Bio X Cell, Inc., West Lebanon, New Hampshire, USA). A number of commercially available anti-PD-1 antibodies are known to those skilled in the art.

In one embodiment, the PD-1 inhibitor is an antibody disclosed in US Patent No. 8354509 or US Patent Application Publications Nos. 2010/0266617 A1, 2013/0108651 A1, 2013/0109843 A2, the disclosure of which is incorporated herein by reference. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody described in US Patent Nos. 8287856, 8580247 and 8168757 and US Patent Application Publications Nos. 2009/0028857 A1, 2010/0285013 A1, 2013/0022600 A1 and 2011/0008369 A1, the contents of which are incorporated herein by reference. In another embodiment, the PD-1 inhibitor is an anti-PD-1 antibody disclosed in US Pat. No. 8,735,553 B1, the disclosure of which is incorporated herein by reference. In one embodiment, the PD-1 inhibitor is pidilizumab, also known as CT-011, which is described in US Pat. No. 8,686,119, the disclosure of which is incorporated herein by reference.

In one embodiment, the PD-1 inhibitor may be a small molecule or a peptide or peptide derivative, such as those described in US Patent Nos. 8,907,053, 9,096,642, and 9,044,442 and US Patent Application Publication No. US 2015/0087581; 1,2,4oxadiazole compounds and derivatives such as those described in US Patent Application Publication No. 2015/0073024; cyclic peptidomimetic compounds and derivatives, such as those described in US Patent Application Publication No. US 2015/0073042; cyclic compounds and derivatives such as those described in US Patent Application Publication No. US 2015/0125491; compounds and derivatives of 1,3,4-oxadiazole and 1,3,4thiadiazole, such as those described in International Patent Application Publication No. WO 2015/033301; peptide-based compounds and derivatives, such as those described in International Patent Application Publications No. WO 2015/036927 and WO 2015/04490, or macrocyclic peptide-based compounds and derivatives, such as those described in US Patent Application Publication No. US 2014/0294898 ; the description of each of which is incorporated herein by reference in its entirety. In one embodiment, the PD-1 inhibitor is cemiplimab, commercially available from Regeneron, Inc.

In one embodiment, the PD-L1 or PD-L2 inhibitor may be any PD-L1 or PD-L2 inhibitor, antagonist, or blocker known in the art. In particular, it is one of the PD-L1 or PD-L2 inhibitors, antagonists or blockers described in more detail in the following paragraphs. The terms inhibitor, antagonist and blocker are used interchangeably herein in relation to PD-L1 and PD-L2 inhibitors. For the avoidance of doubt, references to

- 117 045580 herein for a PD-L1 or PD-L2 inhibitor, which is an antibody, may refer to a compound or antigen-binding fragments, variants, conjugates or biosimilars thereof. For the avoidance of doubt, references herein to a PD-L1 or PD-L2 inhibitor may refer to the compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

In some embodiments, the compositions, processes and methods described herein include a PD-L1 or PD-L2 inhibitor. In some embodiments, the PD-L1 or PD-L2 inhibitor is a small molecule. In a preferred embodiment, the PD-L1 or PD-L2 inhibitor is an antibody (ie, an anti-PD-1 antibody), a fragment thereof, including Fab fragments, or a single chain variable fragment (scFv) thereof. In some embodiments, the PD-L1 or PD-L2 inhibitor is a polyclonal antibody. In a preferred embodiment, the PD-L1 or PD-L2 inhibitor is a monoclonal antibody. In some embodiments, the PD-L1 or PD-L2 inhibitor competes for binding with PD-L1 or PD-L2 and/or binds to an epitope on PD-L1 or PD-L2. In one embodiment, the antibody competes for binding to PD-L1 or PD-L2 and/or binds to an epitope on PD-L1 or PD-L2.

In some embodiments, the PD-L1 inhibitors provided herein are selective for PD-L1 because the compounds bind or interact with PD-L1 at significantly lower concentrations than they bind or interact with other receptors, including the PD-L1 receptor. L2. In some embodiments, the compounds bind to the PD-L1 receptor with a binding constant that is at least about 2 times the concentration, about 3 times the concentration, about 5 times the concentration, about 10 times the concentration, about 20 times the concentration, approximately 30 times the concentration, approximately 50 times the concentration, approximately 100 times the concentration, approximately 200 times the concentration, approximately 300 times the concentration, or approximately 500 times the concentration than the receptor PD-L2.

In some embodiments, the PD-L2 inhibitors provided herein are selective for PD-L2 because the compounds bind or interact with PD-L2 at significantly lower concentrations than they bind or interact with other receptors, including the PD-L2 receptor. L1. In some embodiments, the compounds bind to the PD-L2 receptor with a binding constant that is at least about 2 times the concentration, about 3 times the concentration, about 5 times the concentration, about 10 times the concentration, about 20 times the concentration, approximately 30 times the concentration, approximately 50 times the concentration, approximately 100 times the concentration, approximately 200 times the concentration, approximately 300 times the concentration, or approximately 500 times the concentration than the receptor PD-L1.

Without being limited to any theory, it is believed that tumor cells express PD-L1 and that T cells express PD-1. However, expression of PD-L1 by tumor cells is not required for the effectiveness of PD-1 or PD-L1 inhibitors or blockers. In one embodiment, the tumor cells express PD-L1. In another embodiment, the tumor cells do not express PD-L1. In some embodiments, the methods may include a combination of PD-1 and a PD-L1 antibody, such as those described herein, in combination with a TIL. Administration of the combination of PD-1 and PD-L1 antibody and TILs can be simultaneous or sequential.

In some embodiments, a PD-L1 and/or PD-L2 inhibitor is an inhibitor that binds human PD-L1 and/or PD-L2 with a KD of about 100 pM or less, binds human PD-L1 and/or PD-L2 to KD of about 90 pM or less, binds human PD-L1 and/or PD-L2 with a KD of about 80 pM or less, binds human PD-L1 and/or PD-L2 with a KD of about 70 pM or less, binds PD-L1 and /or human PD-L2 with a KD of about 60 pM or less, a KD of about 50 pM or less, binds human PD-L1 and/or PD-L2 with a KD of about 40 pM or less, or binds PD-L1 and/or PD- Human L2 with a KD of about 30 pM or less.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is an inhibitor that binds to human PD-L1 and/or PD-L2 with a kassoc of about 7.5 x 105 l/ms or faster, binds to PD-L1 and/or or human PD-L2 with a kassoc of about 8x105 l/ms or faster, binds to human PD-L1 and/or PD-L2 with a kassoc of about 8.5x105 l/ms or faster, binds to PD-L1 and/or PD-L2 human kassoc about 9x105 l/ms or faster, binds to human PD-L1 and/or PD-L2 with kassoc about 9.5x105 l/ms and/or faster, or binds to human PD-L1 and/or PD-L2 with kassoc about 1x106 l/ms or faster.

In some embodiments, the PD-L1 and/or PD-L2 inhibitor is an inhibitor that binds to human PD-L1 or PD-L2 with a kdissoc of about 2x10-5 h/s or slower, binds to PD-1 human, with a kdissoc of about 2.1x10 ^-5 l/s or slower, binds to human PD-1 with a kdissoc of about 2.2x10 ^-5 l/s or slower, binds to human PD-1 with a kdissoc of about 2.3x10 ^-5 l/s or slower, binds to human PD-1 with a kdissoc of about 2.4x10 ^-5 l/s or slower, binds to human PD-1 with a kdissoc of about 2.5x10 ^-5 l/s or slower, binds to PD- 1 person with a kdissoc of about 2.6x10 ^-5 l/s or slower, bound to PD-L1 or PD-L2 person with a kdissoc of about 2.7x10 ^-5 l/s or slower, or bound to PD-L1 or PD-L2 a person with kdissoc is about 3x10 ^-5 l/s or slower.

In some embodiments, a PD-L1 and/or PD-L2 inhibitor is an inhibitor that blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 10 nM or less; blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 9 nM or less; blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 8 nM or less; blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 7 nM or less; blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 6 nM or less; blocks or inhibits the binding of human PDL1 or PD-L2 to human PD-1 with an IC ₅₀ of about 5 nM or less; blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 4 nM or less; blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 3 nM or less; blocks or inhibits the binding of human PD-L1 or PD-L2 to human PD-1 with an IC ₅₀ of about 2 nM or less; or blocks human PD-1, or blocks binding of human PD-L1 or PD-L2 to human PD-1 with an IC50 of about 1 nM or less.

In one embodiment, the PD-L1 inhibitor is durvalumab, also known as MEDI4736 (which is commercially available from Medimmune, LLC, Gaithersburg, MD, a subsidiary of AstraZeneca plc.), or antigen binding fragments, conjugates or variants thereof. In one embodiment, the PD-L1 inhibitor is an antibody disclosed in US Patent No. 8,779,108 or US Patent Application Publication No. 2013/0034559, the disclosure of which is incorporated herein by reference. The clinical efficacy of durvalumab is described in Page, et al., Ann. Rev. Med., 2014, 65, 185-202; Brahmer, et al., J. Clin. Oncol. 2014, 32, 5s (supplement, abstract 8021); and McDermott, et al., Cancer Treatment Rev., 2014, 40, 1056-64. The preparation and properties of durvalumab are described in US Pat. No. 8,779,108, the disclosure of which is incorporated herein by reference. The amino acid sequences of durvalumab are presented in Table. 20. The monoclonal antibody durvalumab includes disulfide bonds at positions 22-96, 22-96, 23'-89', 23'-89', 135'-195', 135'-195', 148-204, 148-204, 215'-224, 215'-224, 230-230, 233-233, 265-325, 265325, 371-429 and 371-429'; and N-glycosylation sites at positions Asn-301 and Asn-301.

In one embodiment, the PD-L1 inhibitor comprises a heavy chain represented by SEQ ID NO: 178 and a light chain represented by SEQ ID NO: 179. In one embodiment, the PD-L1 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In one embodiment, the PD-L1 inhibitor contains a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In one embodiment, the PD-L1 inhibitor contains a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively. In one embodiment, the PD-L1 inhibitor contains a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively.

In one embodiment, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of durvalumab. In one embodiment, the heavy chain variable region (VH) of the PD-L1 inhibitor contains the sequence shown in SEQ ID NO: 180, and the light chain variable region ( _VL ) of the PD-L1 inhibitor contains the sequence shown in SEQ ID NO: 181. or their conservative amino acid substitutions. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 180 and SEQ ID NO: 181, respectively. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 180 and SEQ ID NO: 181, respectively. In one embodiment, the PD-L1 inhibitor contains V _H and V _L regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 180 and SEQ ID NO: 181, respectively. In one embodiment, the PD-L1 inhibitor comprises V _H and V _L regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 180 and SEQ ID

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NO: 181 respectively. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO:

180 and SEQ ID NO: 181, respectively.

In one embodiment, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 182, SEQ ID NO: 183 and SEQ ID NO: 184, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 185, SEQ ID NO: 186 and SEQ ID NO: 187, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on PD-L1 as any of the above antibodies.

In one embodiment, the PD-L1 inhibitor is a biosimilar anti-PD-L1 monoclonal antibody approved by drug regulatory authorities for durvalumab. In one embodiment, the biosimilar comprises an anti-PD-L1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is durvalumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-L1 antibody authorized or submitted for registration, wherein the anti-PD-L1 antibody is provided in a formulation that is different from the formulations of the reference drug or reference biological product, wherein the reference drug or reference biological product is is durvalumab. An anti-PDL1 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is durvalumab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is durvalumab.

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Table 20

Amino acid sequences for PD-L1 inhibitors related to durvalumab

Identifier Sequence (single letter amino acid symbols) SEQIDNO:178 heavy chain durvalumab EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVANIKQDGSEKYY 60 VDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDY WGQGTLVTVS 120 SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS 180 SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPEFEG 240 GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300 NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPASIEKTI SKAKGQPREP QVYTLPPSRE 360 EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR 420 WQQGNVFSCS VMHEALHNHY TQKSLSLSPGK 451 SEQIDNO:179 durvalumab light chain EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVAN EIVLTQSPGT 60 LSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIY DASSRATGIP DRFSGSGSGT 120 DFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIKRT VAAPSVFIFP PSDEQLKSGT 180 ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH 240 KVYACEVTHQ GLSSPVTKSF NRGEC 265

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SEQIDNO:180 variable heavy chain durvalumab EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVANIKQDGSEKYY 60 VDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDY WGQGTLVTVS 120 S 121 SEQIDNO:181 durvalumab variable light chain EIVLTQSPGT LSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIY DASSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIK 108 SEQIDNO:182 CDR1 severe durvalumab chains RYWMS 5 SEQIDNO:183 CDR2 severe chains durvalumab NIKQDGSEKY YVDSVKG 17 SEQIDNO:184 CDR3 severe durvalumab chains EGGWFGELAF DY 12 SEQIDNO:185 CDR1 light chains durvalumab RASQRVSSSY LA 12 SEQIDNO:186 CDR2 easy durvalumab chains DASSRAT 7 SEQIDNO:187 CDR3 easy durvalumab chains QQYGSLPWT 9

In one embodiment, the PD-L1 inhibitor is avelumab, also known as MSB0010718C (commercially available from Merck KGaA/EMD Serono), or antigen binding fragments thereof:, conjugates: or variants:. The preparation and properties of avelumab are described in US Patent Application Publication No. US 2014/0341917 A1, the disclosure of which is specifically incorporated herein by reference. The amino acid sequences of avelumab are presented in table. 21. Avelumab has disulfide bonds within the heavy chain (C23-C104) at positions 22-96, 147-203, 264-324, 370-428, 22-96, 147-203, 264-324 and 370-428; disulfide bonds within the light chain (C23-C104) positions. 22'-90', 138'-197', 22'-90' and 138'197'; disulfide bonds within the light chain (h 5-CL 126) at positions 223-215' and 223-215'; disulfide bonds within the heavy chain (h 11, h 14) at positions 229-229 and 232-232; sites: N-glycosylation (H CH2 N84.4) at positions 300, 300; fucosylated complex biantennary glycans: CHO-type; and cleavage of the C-terminal lysine H of CHS K2 at positions 450 and 450'.

- 122 045580

In one embodiment, the PD-L1 inhibitor comprises a heavy chain represented by SEQ ID NO: 188 and a light chain represented by SEQ ID NO: 189. In one embodiment, the PD-L1 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 188 and SEQ ID NO: 189, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In one embodiment, the PD-L1 inhibitor contains a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 188 and SEQ ID NO: 189, respectively. In one embodiment, the PD-L1 inhibitor contains a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 188 and SEQ ID NO: 189, respectively.

In one embodiment, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of avelumab. In one embodiment, the heavy chain variable region (VH) of the PD-L1 inhibitor contains the sequence shown in SEQ ID NO: 190, and the light chain variable region ( _VL ) of the PD-L1 inhibitor contains the sequence shown in SEQ ID NO: 191. or their conservative amino acid substitutions. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In one embodiment, the PD-L1 inhibitor contains V _H and V _L regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In one embodiment, the PD-L1 inhibitor contains V _H and V _L regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 190 and SEQ ID NO: 191, respectively. In one embodiment, the PD-L1 inhibitor comprises V _H and V _L regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 190 and SEQ ID NO: 191, respectively.

In one embodiment, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 192, SEQ ID NO: 193 and SEQ ID NO: 194, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 195, SEQ ID NO: 196 and SEQ ID NO: 197, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on PD-L1 as any of the above antibodies.

In one embodiment, the PD-L1 inhibitor is a biosimilar anti-PD-L1 monoclonal antibody approved by drug regulatory authorities for avelumab. In one embodiment, the biosimilar comprises an anti-PD-L1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is avelumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-L1 antibody authorized or submitted for registration, wherein the anti-PD-L1 antibody is provided in a formulation that is different from the formulations of the reference drug or reference biological product, wherein the reference drug or reference biological product is avelumab. An anti-PD-L1 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is avelumab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients

- 123 045580 substances are the same or different from the excipients contained in the reference medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is avelumab.

Table 21

Amino acid sequences for PD-L1 inhibitors associated with avelumab

Identifier Sequence (single letter amino acid symbols) SEQIDNO:188 avelumab heavy chain EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSS IYPSGGITFY 60 ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYW GQGTLVTVSS 120 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS 180 GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG 240 PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360 LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQIDNO:189 avelumab light chain QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV 60 SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVL GQPKANPTTVT 120 LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSK QSNNKYAASS 180

- 124 045580

YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS 216 SEQIDNO:190 durvalumab light chain EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSS IYPSGGITFY 60 ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYW GQGTLVTVSS 120 SEQIDNO:191 avelumab variable light chain QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMI YDVSNRPSGV 60 SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVL 110 SEQIDNO:192 CDR1 of avelumab light chain SYIMM 5 SEQIDNO:193 CDR2 of avelumab heavy chain SIYPSGGITF YADTVKG 17 SEQIDNO:194 CDR3 of avelumab heavy chain IKLGTVTTVD Y 11 SEQIDNO:195 CDR1 of avelumab light chain TGTSSDVGGYNYVS 14 SEQIDNO:196 CDR2 of avelumab light chain DVSNRPS 7 SEQIDNO:197 CDR3 of avelumab light chain SSYTSSSTRV 10

In one embodiment, the PD-L1 inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (commercially available as TECENTRIQ from Genentech, Inc., a subsidiary of Roche Holding AG, Basel, Switzerland), or antigen binding fragments, conjugates, or variants thereof. In one embodiment, the PD-L1 inhibitor is an antibody described in US Pat. No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In one embodiment, the PD-L1 inhibitor is an antibody described in US Patent Application Publication Nos. 2010/0203056 A1, 2013/0045200 A1, 2013/0045201 A1, 2013/0045202 A1, or 2014/0065135 A1, the disclosure of which is specifically included in this document by reference. The preparation and properties of atezolizumab are described in US Pat. No. 8,217,149, the disclosure of which is incorporated herein by reference. The amino acid sequences of atezolizumab are presented in table. 22. Atezolizumab has disulfide bonds within the heavy chain (C23-C104) at positions 22-96, 145-201, 262-322, 368-426, 22-96, 145-201, 262-322 and 368-426; disulfide bonds within the light chain (C23-C104) at positions 23'-88', 134'194', 23'-88' and 134'-194'; disulfide bonds within the light chain (h 5-CL 126) at positions 221-214' and 221-214'; disulfide bonds within the heavy chain (h 11, h 14) at positions 227-227 and 230-230; and N-glycosylation sites (H CH2 N84.4>A) at positions 298 and 298'.

In one embodiment, the PD-L1 inhibitor comprises a heavy chain represented by SEQ ID NO: 198 and a light chain represented by SEQ ID NO: 199. In one embodiment, the PD-L1 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 198 and SEQ ID NO: 199, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants thereof or conjugates. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In one embodiment, the PD-L1 inhibitor contains a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In one embodiment, the PD-L1 inhibitor contains a heavy and a light chain, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 198 and SEQ ID NO: 199, respectively. In one embodiment, the PD-L1 inhibitor comprises a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 198 and SEQ ID NO: 199, respectively.

In one embodiment, the PD-L1 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of atezolizumab. In one embodiment, the heavy chain variable region (VH) of the PD-L1 inhibitor contains the sequence shown in SEQ ID NO: 200, and the light chain variable region ( _VL ) of the PD-L1 inhibitor contains the sequence shown in SEQ ID NO: 201. or their conservative amino acid substitutions. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 200 and SEQ ID NO: 201, respectively. In one embodiment, the PD-L1 inhibitor contains VH and VL regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 200 and SEQ ID NO: 201, respectively.

In one embodiment, the PD-L1 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 202, SEQ ID NO: 203 and SEQ ID NO: 204, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 205, SEQ ID NO: 206 and SEQ ID NO: 207, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on PD-L1 as any of the above antibodies.

In one embodiment, the anti-PD-L1 antibody is a biosimilar anti-PD-L1 monoclonal antibody approved by drug regulatory authorities for atezolizumab. In one embodiment, the biosimilar comprises an anti-PD-L1 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is atezolizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an anti-PD-L1 antibody authorized or submitted for registration, wherein the anti-PD-L1 antibody is provided in a formulation that is different from the formulations of the reference drug or reference biological product, wherein the reference drug or reference biological product is is atezolizumab. An anti-PD-L1 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is atezolizumab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is atezolizumab.

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Table 22

Amino acid sequences for PD-L1 inhibitors associated with atezolizumab

Identifier Sequence (single letter amino acid symbols) SEQIDNO:198 atezolizumab heavy chain EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA PGKGLEWVAW ISPYGGSTYY 60 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSSAS 120 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTCPPC PAPELLGGPS 240 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYAST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 360 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 420 GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 SEQIDNO:199 atezolizumab light chain DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ Ш NO:200 EVQLVESGGG LVQPGGSLRL SCAASGFTFS DSWIHWVRQA

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atezolizumab variable heavy chain PGKGLEWVAW ISPYGGSTYY 60 ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCARRH WPGGFDYWGQ GTLVTVSA 118 SEQIDNO:201 atezolizumab variable light chain DIQMTQSPSS LSASVGDRVT ITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YLYHPATFGQ GTKVEIKR 108 SEQ Ш NO:202 CDR1 severe chains atezolizumab GFTFSDSWIH 10 SEQ GO NO:203 CDR2 severe chains atezolizumab AWISPYGGST YYADSVKG 18 SEQ GO NO:204 CDR3 severe chains atezolizumab RHWPGGFDY 9 SEQ GO NO:205 CDR1 of atezolizumab light chain RASQDVSTAV A 11 SEQ GO NO:206 CDR2 of atezolizumab light chain SASFLYS 7 SEQ GO NO:207 CDR3 of atezolizumab light chain QQYLYHPAT9

In one embodiment, PD-L1 inhibitors include the antibodies described in US Patent Application Publication No. US 2014/0341917 A1, the disclosure of which is incorporated herein by reference. In another embodiment, antibodies that compete with any of these antibodies for binding to PD-L1 are also included. In one embodiment, the anti-PD-L1 antibody is MDX-1105, also known as BMS-935559, which is described in US Pat. No. 7,943,743, the disclosure of which is incorporated herein by reference. In one embodiment, the anti-PD-L1 antibody is selected from the anti-PD-L1 antibodies described in US Pat. No. 7,943,743, which are incorporated herein by reference.

In one embodiment, the PD-L1 inhibitor is a commercially available monoclonal antibody such as INVIVOMAB anti-m-PD-L1 clone 10F.9G2 (catalog number BE0101, Bio X Cell, Inc., West Lebanon, NH, USA ). In one embodiment, the anti-PD-L1 antibody is a commercially available monoclonal antibody such as AFFYMETRIX EBIOSCIENCE (MIH1). A number of commercially available anti-PD-L1 antibodies are known to those skilled in the art.

In one embodiment, the PD-L2 inhibitor is a commercially available monoclonal antibody such as BIOLEGEND 24F.10C12 Mouse IgG2a, isotype k (catalog no. 329602 Biolegend, Inc., San Diego, Calif.), SIGMA anti-PD-L2 antibody. (catalog number SAB3500395, Sigma-Aldrich Co., St. Louis, MO) or other commercially available antibodies

- 128 045580 to PD-L2, known to specialists in the art.

In some embodiments, the present invention includes a method of treating a patient suffering from cancer, comprising the steps of administering a TIL regimen, the TIL regimen comprising a TIL product genetically modified to express a CCR, further comprising the step of administering either a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the present invention includes a composition comprising: (i) a TIL product genetically modified to express a CCR, and (ii) either a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the present invention includes a kit comprising: (i) a TIL product genetically modified to express a CCR, and (ii) either a PD-1 inhibitor or a PD-L1 inhibitor.

3. Combinations with CTLA-4 inhibitors.

In some embodiments, TIL therapy provided to patients suffering from cancer may include treatment with TIL therapeutic populations alone or may include a combination treatment comprising TILs and one or more CTLA-4 inhibitors.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily and is expressed on the surface of T helper cells. CTLA-4 is a negative regulator of CD28-dependent T cell activation and acts as a checkpoint for adaptive immune responses. Like the T cell costimulatory protein CD28, CTLA-4 binding antigen presents CD80 and CD86 on cells. CTLA-4 delivers a suppressive signal to T cells, whereas CD28 delivers a stimulatory signal. Human antibodies to human CTLA-4 are described as immunostimulatory modulators in many disease conditions, such as the treatment or prevention of viral and bacterial infections and the treatment of cancer (WO 01/14424 and WO 00/37504). A number of fully human monoclonal antibodies (mAbs) to human CTLA-4 have been studied in clinical trials for the treatment of various types of solid tumors, including, but not limited to, ipilimumab (MDX-010) and tremelimumab (CP-675206).

In some embodiments, the CTLA-4 inhibitor may be any CTLA-4 inhibitor or CTLA-4 blocker known in the art. Specifically, it is one of the CTLA-4 inhibitors or blockers described in more detail in the following paragraphs. The terms inhibitor, antagonist and blocker are used interchangeably herein with respect to CTLA-4 inhibitors. For the avoidance of doubt, references herein to a CTLA-4 inhibitor that is an antibody may refer to the compound or antigen binding fragments, variants, conjugates or biosimilars thereof. For the avoidance of doubt, references herein to a CTLA-4 inhibitor may also refer to the small molecule compound or a pharmaceutically acceptable salt, ester, solvate, hydrate, co-crystal or prodrug thereof.

Suitable CTLA-4 inhibitors for use in the methods of the present invention include, but are not limited to, anti-CTLA-4 antibodies, anti-human CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies , monoclonal antibodies to CTLA-4, polyclonal antibodies to CTLA-4, chimeric antibodies to CTLA-4, MDX-010 (ipilimumab), tremelimumab, antibodies to CD28, antibodies to CTLA-4 adnectin, domain antibodies to CTLA-4, single chain anti-CTLA-4 fragments, anti-CTLA-4 heavy chain fragments, anti-CTLA-4 light chain fragments, CTLA-4 inhibitors that agonize the co-stimulatory pathway, antibodies described in PCT Publication No. WO 2001/014424, antibodies described in PCT publication No. WO 2004/035607, the antibodies described in US publication No. 2005/0201994, and the antibodies described in issued European patent No. EP. 1212422 B1, the description of each of which is incorporated herein by reference. Additional antibodies to CTLA-4 are described in US patent Nos. 5811097, 5855887, 6051227 and 6984720; in PCT publications No. WO 01/14424 and WO 00/37504; and US Publication Nos. 2002/0039581 and 2002/086014, the description of each of which is incorporated herein by reference. Other anti-CTLA-4 antibodies that can be used in the method of the present invention include, for example, those described in the following publications: WO 98/42752; US Patent Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin. Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998), and US Patent Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281, each of which is incorporated herein by reference.

Additional CTLA-4 inhibitors include, but are not limited to, the following: any inhibitor that is capable of interfering with the ability of the CD28 antigen to bind to its cognate ligand, inhibiting the ability of CTLA-4 to bind to its cognate ligand, enhancing T cell responses through a co-stimulatory pathway, interfering with the ability of B7 to bind with CD28 and/or CTLA-4, disrupt the ability of B7 to activate the costimulatory pathway, disrupt the ability of CD80 to bind to CD28 and/or CTLA-4, disrupt the ability of CD80 to activate the costimulatory pathway, disrupt the ability of CD86 to bind to CD28 and/or CTLA-4, disrupt the ability of CD86 to activate the costimulatory pathway and to disrupt the costimulatory pathway in general from its activation. This necessarily includes small molecule inhibitors of CD28, CD80, CD86, CTLA-4, among other co-stimulatory pathway participants; antibodies directed against CD28, CD80, CD86, CTLA-4, among other members of the cost- 129 045580 pathway; antisense molecules directed against CD28, CD80, CD86, CTLA-4, among other members of the co-stimulatory pathway; adnectins directed against CD28, CD80, CD86, CTLA-4, among other members of the costimulatory pathway, RNAi inhibitors (both single-stranded and double-stranded) CD28, CD80, CD86, CTLA-4, among other members of the costimulatory pathway, among other CTLA inhibitors -4.

In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 with a K _d of about 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 or less, 10 ^-9 M or less, 10 ^-10 M or less , 10 ^-11 M or less, 10 ^-12 M or less, for example, from 10 ^-13 M to 10 ^-16 M, or within any range having any two of the above values as endpoints. In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 with a K _d no more than 10 times greater than that of ipilimumab when compared using the same assay. In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 with a Kd that is approximately the same or less (e.g., up to 10 times less or up to 100 times less) than ipilimumab when compared using the same assay. In some embodiments, the IC50 values for the CTLA-4 inhibitor's inhibition of CTLA-4 binding to CD80 or CD86 are no more than 10-fold greater than those for ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively, when compared using the same assay. In some embodiments, the IC ₅₀ values for the CTLA-4 inhibitor inhibition of CTLA-4 binding to CD80 or CD86 are about the same as or less (e.g., up to 10-fold less or up to 100-fold less) than ipilimumab-mediated inhibition of CTLA-4 binding to CD80 or CD86, respectively, when compared using the same assay.

In some embodiments, the CTLA-4 inhibitor is used in an amount sufficient to inhibit the expression and/or reduce the biological activity of CTLA-4 by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a suitable control, for example 50% to 75%, 75% to 90% or 90% to 100%. In some embodiments, a CTLA-4 pathway inhibitor is used in an amount sufficient to reduce the biological activity of CTLA-4 by reducing the binding of CTLA-4 to CD80, CD86, or both by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a suitable control, for example, from 50% to 75%, from 75% to 90% or from 90% to 100% relative to a suitable control. A suitable control in the context of assessing or quantifying the effect of an agent of interest is usually a comparable biological system (e.g., cells or subject) that has not been exposed to or treated with the agent of interest, such as a CTLA-4 pathway inhibitor (or has been exposed or treated in negligible amounts) . In some embodiments, the biological system may serve as its own control (e.g., the biological system may be assessed before exposure or treatment to an agent and compared to the state after the exposure or treatment begins or ends. In some embodiments, a historical control may be used.

In one embodiment, the CTLA-4 inhibitor is ipilimumab (commercially available as Yervoy from Bristol-Myers Squibb Co.) or biosimilars, antigen binding fragments, conjugates or variants thereof. As is known in the art, ipilimumab refers to an anti-CTLA-4 antibody, a fully human IgGK antibody derived from a transgenic mouse with human genes encoding the heavy and light chains to create a functional human repertoire. Ipilimumab may also be referred to by CAS Registration Number 477202-00-9 and PCT publication WO 01/14424, which is incorporated herein by reference in its entirety for all purposes. It is described as antibody 10DI. Specifically, ipilimumab comprises a light chain variable region and a heavy chain variable region (with a light chain variable region comprising SEQ ID NO: 211 and a heavy chain variable region comprising SEQ ID NO: 210). The pharmaceutical composition of ipilimumab includes all pharmaceutically acceptable compositions containing ipilimumab and one or more diluents, carriers or excipients. An example of a pharmaceutical composition containing ipilimumab is described in International Patent Application Publication No. WO 2007/67959. Ipilimumab can be given intravenously (IV).

In one embodiment, the CTLA-4 inhibitor comprises a heavy chain represented by SEQ ID NO: 208 and a light chain represented by SEQ ID NO: 209. In one embodiment, the CTLA-4 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 208 and SEQ ID NO: 209, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In one embodiment, the CTLA-4 inhibitor comprises

- 130 045580 heavy and light chains, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 208 and SEQ ID NO: 209, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 208 and SEQ ID NO: 209, respectively.

In one embodiment, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of ipilimumab. In one embodiment, the heavy chain variable region (VH) of the CTLA-4 inhibitor contains the sequence shown in SEQ ID NO: 210, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor contains the sequence shown in SEQ ID NO: 211. or their conservative amino acid substitutions. In one embodiment, the CTLA-4 inhibitor contains VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 210 and SEQ ID NO: 211, respectively. In one embodiment, the CTLA-4 inhibitor contains VH and _VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 210 and SEQ ID NO: 211, respectively. In one embodiment, the CTLA-4 inhibitor contains VH and _VL regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 210 and SEQ ID NO: 211, respectively. In one embodiment, the CTLA-4 inhibitor contains VH and _VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 210 and SEQ ID NO: 211, respectively. In one embodiment, the CTLA-4 inhibitor contains V _H and V _L regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 210 and SEQ ID NO: 211, respectively.

In one embodiment, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 212, SEQ ID NO: 213 and SEQ ID NO: 214, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 215, SEQ ID NO: 216 and SEQ ID NO: 217, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on CTLA-4 as any of the above antibodies.

In one embodiment, the CTLA-4 inhibitor is a biosimilar CTLA-4 monoclonal antibody approved by drug regulatory authorities for ipilimumab. In one embodiment, the biosimilar comprises an anti-CTLA-4 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is ipilimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequences of ipilimumab are presented in table. 23. In some embodiments, the biosimilar is an anti-CTLA-4 antibody authorized or submitted for registration, wherein the anti-CTLA-4 antibody is provided in a formulation that is different from the compositions of the reference drug or reference biological product, wherein the reference drug or reference biological the product is ipilimumab. An anti-CTLA-4 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is ipilimumab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is ipilimumab.

- 131 045580

Table 23

Amino acid sequences of ipilimumab

Identifier Sequence (single letter amino acid symbols) SEQ Ш NO:208 heavy chain ipilimumab QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGNNKYY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSSAS 120 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTH 225 SEQ Ш NO:209 light chain EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIP 60

- 132 045580

ipilimumab DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIKRT VAAPSVFIFP 120 PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSKDSTYSLSSTL 180 TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC 215 SEQIDNO:210 variable heavy chain ipilimumab QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYTMHWVRQA PGKGLEWVTF ISYDGNNKYY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAIYYCARTG WLGPFDYWGQ GTLVTVSS 118 SEQIDNO:211 variable light chain ipilimumab EIVLTQSPGT LSLSPGERAT LSCRASQSVG SSYLAWYQQK PGQAPRLLIY GAFSRATGIP 60 DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPWTFG QGTKVEIK 108 SEQIDNO:212 CDR1 severe chains ipilimumab GFTFSSYT 8 SEQIDNO:213 CDR2 severe chains ipilimumab TFISYDGNNK 10 SEQIDNO:214 CDR3 severe chains ipilimumab ARTGWLGPFD Y 11 SEQIDNO:215 CDR1 light chains ipilimumab QSVGSSY 7 SEQIDNO:216 CDR2 light chains ipilimumab GAF 3 SEQIDNO:217 CDR3 easy chains ipilimumab QQYGSSPWT 9

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2 .5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg,

- 133 045580 about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg /kg or about 10 mg/kg. In some embodiments, administration of ipilimumab is started at 1.

2, 3, 4, or 5 weeks before resection (ie, until a tumor sample is obtained from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg or about 500 mg. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat unresectable or metastatic melanoma. In some embodiments, ipilimumab is administered for the treatment of unresectable or metastatic melanoma at a dose of about mg/kg every 3 weeks for a maximum of 4 doses. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered for the adjuvant treatment of melanoma. In some embodiments, ipilimumab is administered for the adjuvant treatment of melanoma at a dose of about 10 mg/kg every 3 weeks for 4 doses, and then 10 mg/kg every 12 weeks for up to 3 years. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat advanced renal cell carcinoma. In some embodiments, ipilimumab is administered for the treatment of advanced renal cell carcinoma at a dose of about 1 mg/kg immediately following administration of 3 mg/kg nivolumab on the same day, every 3 weeks for 4 doses. In some embodiments, after administration of 4 doses of the combination, nivolumab may be administered as a single agent according to standard dosing regimens for the treatment of advanced renal cell carcinoma and/or renal cell carcinoma. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR). In some embodiments, ipilimumab is administered for the treatment of metastatic colorectal cancer with microsatellite instability high (MSI-H) or mismatch repair deficiency (dMMR) at a dose of about 1 mg/kg IV over 30 minutes immediately after nivolumab at a dose of 3 mg/kg IV at for 30 minutes on the same day, every 3 weeks in 4 doses. In some embodiments, after administration of 4 doses of the combination, nivolumab is administered as a single agent as recommended according to standard dosing regimens for the treatment of microsatellite instability-high (MSI-H) or mismatch repair defective (dMMR) metastatic colorectal cancer. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat hepatocellular carcinoma. In some embodiments, ipilimumab is administered for the treatment of hepatocellular carcinoma at a dose of about 3 mg/kg IV over 30 minutes immediately after administration of nivolumab at a dose of 1 mg/kg IV over 30 minutes on the same day, every 3 weeks for 4 doses. In some embodiments, after administration of 4 doses of the combination, nivolumab is administered as a single agent in

- 134 045580 in accordance with standard dosing regimens for the treatment of hepatocellular carcinoma. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat metastatic non-small cell lung cancer. In some embodiments, ipilimumab is administered for the treatment of metastatic non-small cell lung cancer at a dose of about 1 mg/kg every 6 weeks with nivolumab at a dose of 3 mg/kg every 2 weeks. In some embodiments, ipilimumab is administered for the treatment of metastatic non-small cell lung cancer at a dose of about 1 mg/kg every 6 weeks with nivolumab at a dose of 360 mg every 3 weeks and 2 cycles of platinum doublet chemotherapy. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, ipilimumab is administered to treat malignant pleural mesothelioma. In some embodiments, ipilimumab is administered for the treatment of malignant pleural mesothelioma at a dose of about 1 mg/kg every 6 weeks with nivolumab at a dose of 360 mg every 3 weeks. In some embodiments, ipilimumab administration begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, ipilimumab administration begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

Tremelimumab (also known as CP-675206) is a fully human IgG2 monoclonal antibody and has CAS number 745013-59-6. Tremelimumab is described as antibody 11.2.1 in US Pat. No. 6,682,736 (incorporated herein by reference). The amino acid sequences of the heavy chain and light chain of tremelimumab are shown in SEQ ID NO: 218 and 219, respectively. Tremelimumab has been studied in clinical trials for the treatment of various tumors, including melanoma and breast cancer; in which tremelimumab was administered intravenously as single or multiple doses every 4 or 12 weeks in a dose range of 0.01 to 15 mg/kg. In the regimens provided by the present invention, tremelimumab is administered topically, in particular intradermally or subcutaneously. The effective amount of tremelimumab administered intradermally or subcutaneously is usually in the range of 5-200 mg/dose per person. In some embodiments, the effective amount of tremelimumab is in the range of 10-150 mg/dose per person per dose. In some specific embodiments, the effective amount of tremelimumab is about 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175, or 200 mg/dose per person.

In one embodiment, the CTLA-4 inhibitor comprises a heavy chain represented by SEQ ID NO: 218 and a light chain represented by SEQ ID NO: 219. In one embodiment, the CTLA-4 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 218 and SEQ ID NO: 219, respectively.

In one embodiment, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of tremelimumab. In one embodiment, the heavy chain variable region (VH) of the CTLA-4 inhibitor contains the sequence shown in SEQ ID NO: 220, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor contains the sequence shown in SEQ ID NO: 221. or their conservative amino acid substitutions. In one embodiment, the CTLA-4 inhibitor contains VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 220 and SEQ ID NO: 221, respectively. In one embodiment, the CTLA-4 inhibitor contains VH and VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 220 and SEQ ID NO: 221, respectively. In one embodiment, the CTLA-4 inhibitor contains V _H and V _L regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 220 and SEQ ID NO: 221

- 135 045580 respectively. In one embodiment, the CTLA-4 inhibitor contains VH and VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 220 and

SEQ ID NO: 221 respectively. In one embodiment, the CTLA-4 inhibitor comprises regions

VH and VL, each of which is at least 95% identical to the sequences shown in

SEQ ID NO: 220 and SEQ ID NO: 221 respectively.

In one embodiment, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 222, SEQ ID NO: 223 and SEQ ID NO: 224, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 225, SEQ ID NO: 226 and SEQ ID NO: 227, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on CTLA-4 as any of the above antibodies.

In one embodiment, the CTLA-4 inhibitor is a biosimilar anti-CTLA-4 monoclonal antibody approved by drug regulatory authorities for tremelimumab. In one embodiment, the biosimilar comprises an anti-CTLA-4 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is tremelimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequences of tremelimumab are presented in table. 24. In some embodiments, the biosimilar is an anti-CTLA-4 antibody authorized or submitted for registration, wherein the anti-CTLA-4 antibody is provided in a formulation that is different from the compositions of the reference drug or reference biological product, wherein the reference drug or reference biological the product is tremelimumab. An anti-CTLA-4 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is tremelimumab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product the biological product is tremelimumab.

- 136 045580

Table 24

Tremelimumab amino acid sequences

Identifier Sequence (single letter amino acid symbols) SEQIDNO:218 heavy chain tremelimumab QVQLVESGGG VVQPGRSLRL SCAASGFTFS SYGMHWVRQA PGKGLEWVAVIWYDGSNKYY 60 ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDP RGATLYYYYY GMDVWGQGTT 120 VTVSSASTKG PSVFPLAPCS RSTSESTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA 180 VLQSSGLYSL SSVVTVPSSN FGTQTYTCNV DHKPSNTKVD KTVERKCCVE CPPCPAPPVA 240 GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVQFN WYVDGVEVHN AKTKPREEQF 300 NSTFRVVSVL TVVHQDWLNG KEYKCKVSNK GLPAPIEKTI SKTKGQPREP QVYTLPPSRE 360 EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP MLDSDGSFFL YSKLTVDKSR 420 WQQGNVFSCS VMHEALHNHY TQKSLSLSPGK 451 SEQIDNO:219 light chain tremelimumab DIQMTQSPSS LSASVGDRVT ITCRASQSIN SYLDWYQQKP GKAPKLLIYA ASSLQSGVPS 60 RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YYSTPFTFGP GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ Ш NO:220 variable heavy chain tremelimumab GVVQPGRSLR LSCAASGFTF SSYGMHWVRQ APGKGLEWVA VIWYDGSNKY YADSVKGRFT 60 ISRDNSKNTL YLQMNSLRAE DTAVYYCARD PRGATLYYYY YGMDVWGQGT TVTVSSASTK 120 GPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVH 167

- 137 045580

SEQIDNO:221 variable light chain tremelimumab PSSLSASVGD RVTITCRASQ SINSYLDWYQ QKPGKAPKLL IYAASSLQSG VPSRFSGSGS 60 GTDFTLTISS LQPEDFATYY CQQYYSTPFT FGPGTKVEIK RTVAAPSVFIFPPSDEQLKS 120 GTASVVCLLN NFYPREAKV 139 SEQ GO NO:222 CDR1 heavy chains tremelimumab GFTFSSYGMH 10 SEQ GO NO:223 CDR2 heavy chains tremelimumab VIWYDGSNKY YADSV 15 SEQ GO NO:224 CDR3 severe chains tremelimumab DPRGATLYYY YYGMDV 16 SEQ GO NO:225 Tremelimumab light chain CDR1 RASQSINSYL D 11 SEQ GO NO:226 Tremelimumab light chain CDR2 AASSLQS 7 SEQ GO NO:227 Tremelimumab light chain CDR3 QQYYSTPFT 9

In some embodiments, the CTLA-4 inhibitor is tremelimumab or a biosimilar thereof, wherein tremelimumab is administered at a dose of about 0.5 mg/kg to about 10 mg/kg. In some embodiments, the CTLA-4 inhibitor is tremelimumab or a biosimilar thereof, wherein tremelimumab is administered at a dose of about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2 .5 mg/kg, about 3 mg/kg, about 3.5 mg/kg, about 4 mg/kg, about 4.5 mg/kg, about 5 mg/kg, about 5.5 mg/kg, about 6 mg/kg, about 6.5 mg/kg, about 7 mg/kg, about 7.5 mg/kg, about 8 mg/kg, about 8.5 mg/kg, about 9 mg/kg, about 9.5 mg/kg or about 10 mg/kg. In some embodiments, administration of tremelimumab begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, administration of tremelimumab begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is tremelimumab or a biosimilar thereof, wherein tremelimumab is administered at a dose of about 200 mg to about 500 mg. In some embodiments, the CTLA-4 inhibitor is ipilimumab or a biosimilar thereof, wherein ipilimumab is administered at a dose of about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 320 mg, about 340 mg, about 360 mg, about 380 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg or about 500 mg. In some embodiments, administration of tremelimumab begins 1, 2, 3, 4, or 5 weeks before resection (ie, before obtaining a tumor sample from the subject or patient). In some embodiments, administration of tremelimumab begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In some embodiments, the CTLA-4 inhibitor is tremelimumab or a biosimilar thereof, wherein tremelimumab is administered every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, tremelimumab administration is initiated 1, 2, 3, 4, or 5 weeks prior to resection (ie, prior to obtaining a tumor sample from the subject or patient). In some embodiments, administration of tremelimumab begins 1, 2, or 3 weeks before resection (ie, before obtaining a tumor sample from the subject or patient).

In one embodiment, the CTLA-4 inhibitor is zalifelimab from Agenus or its biosimilars, antigen binding fragments, conjugates, or variants thereof. Zalifrelimab is a fully human monoclonal antibody. Zalifrelimab has a Chemical Abstracts Service (CAS) registration number of 2148321-69-9 and is also known as AGEN1884. The preparation and properties of zalifrelimab are described in US Patent No. 10144779 and US Patent Application Publication No. US2020/0024350 A1, the disclosure of which is incorporated herein by reference.

In one embodiment, the CTLA-4 inhibitor comprises a heavy chain represented by SEQ ID NO: 228 and a light chain represented by SEQ ID NO: 229. In one embodiment, the CTLA-4 inhibitor comprises heavy and light chains having the sequences shown in SEQ ID NO: 228 and SEQ ID NO: 229, respectively, or antigen binding fragments, Fab fragments, single chain variable fragments (scFv), variants or conjugates thereof. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 228 and SEQ ID NO: 229, respectively. In one embodiment, the CTLA-4 inhibitor contains a heavy and a light chain, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 228 and SEQ ID NO: 229, respectively.

In one embodiment, the CTLA-4 inhibitor comprises the heavy and light chain CDRs or variable regions (VRs) of zalifrelimab. In one embodiment, the heavy chain variable region (VH) of the CTLA-4 inhibitor contains the sequence shown in SEQ ID NO: 230, and the light chain variable region ( _VL ) of the CTLA-4 inhibitor contains the sequence shown in SEQ ID NO: 231. or their conservative amino acid substitutions. In one embodiment, the CTLA-4 inhibitor contains VH and _VL regions, each of which is at least 99% identical to the sequences shown in SEQ ID NO: 230 and SEQ ID NO: 231, respectively. In one embodiment, the CTLA-4 inhibitor contains VH and VL regions, each of which is at least 98% identical to the sequences shown in SEQ ID NO: 230 and SEQ ID NO: 231, respectively. In one embodiment, the CTLA-4 inhibitor contains VH and VL regions, each of which is at least 97% identical to the sequences shown in SEQ ID NO: 230 and SEQ ID NO: 231, respectively. In one embodiment, the CTLA-4 inhibitor contains VH and VL regions, each of which is at least 96% identical to the sequences shown in SEQ ID NO: 230 and SEQ ID NO: 231, respectively. In one embodiment, the CTLA-4 inhibitor contains V _H and V _L regions, each of which is at least 95% identical to the sequences shown in SEQ ID NO: 230 and SEQ ID NO: 231, respectively.

In one embodiment, the CTLA-4 inhibitor comprises heavy chain CDR1, CDR2 and CDR3 domains having the sequences set forth in SEQ ID NO: 231, SEQ ID NO: 233 and SEQ ID NO: 234, respectively, or conservative amino acid substitutions thereof, and CDR1 domains , light chain CDR2 and CDR3 having the sequences set forth in SEQ ID NO: 235, SEQ ID NO: 236 and SEQ ID NO: 237, respectively, or conservative amino acid substitutions thereof. In one embodiment, the antibody competes for binding and/or binds to the same epitope on CTLA-4 as any of the above antibodies.

In one embodiment, the CTLA-4 inhibitor is a biosimilar CTLA-4 monoclonal antibody approved by drug regulatory authorities for zalifrelimab. In one embodiment, the biosimilar comprises an anti-CTLA-4 antibody comprising an amino acid sequence that has at least 97% sequence identity, e.g., 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of a reference drug or reference biological a product that contains one or more post-translational modifications compared to a reference drug or reference biological product, wherein the reference drug or reference biological product is zalifrelimab. In some embodiments, the one or more post-translational modifications are selected from one or more of the following: glycosylation, oxidation, deamidation, and truncation. The amino acid sequences of zalifrelimab are presented in table. 25. In some embodiments, the biosimilar is an approved or submitted anti-CTLA-4 antibody, wherein the anti-CTLA-4 antibody is provided in a formulation that is different from the reference drug formulations.

- 139 045580 of a medicinal product or reference biological product, wherein the reference medicinal product or reference biological product is zalifrelimab. An anti-CTLA-4 antibody may be approved by a drug regulatory authority such as the US FDA and/or the European Union EMA. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product The biological product is zalifrelimab. In some embodiments, the biosimilar is provided as a composition that further contains one or more excipients, wherein the one or more excipients is the same or different from the excipients contained in the reference drug or reference biological product, wherein the reference drug or reference biological product The biological product is zalifrelimab.

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Table 25

Amino acid sequences of zalifrelimab

Identifier Sequence (single letter amino acid symbols) SEQ Ш NO:228 heavy chain zalifrelimab EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISSSSSYIYY 60 ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVG LMGPFDIWGQ GTMVTVSSAS 120 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180 YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPELLGGPS 240 VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 300 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 360 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 420 GNVFSCSVMH EALHNHYTQK SLSLSPGK 448 SEQ Ш NO:229 light chain zalifrelimab EIVLTQSPGT LSLSPGERAT LSCRASQSVS RYLGWYQQKP GQAPRLLIYG ASTRATGIPD 60 RFSGSGSGTD FTLTITRLEP EDFAVYYCQQ YGSSPWTFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 SEQ Ш NO:230 variable heavy chain zalifrelimab EVOLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVROA PGKGLEWVSS ISSSSSYIYY 60 ADSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVG LMGPFDIWGQ GTMVTVSS 118 SEQIDNO:231 variable light chain zalifrelimab EIVLTOSPGT LSLSPGERAT LSCRASQSVS RYLGWYQQKP GQAPRLLIYG ASTRATGIPD 60 RFSGSGSGTD FTLTITRLEP EDFAVYYCQQ YGSSPWTFGQ GTKVEIK 107 SEQ Ш NO:232 CDR1 severe chains GFTFSSYS 8

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zalifrelimab SEQ Ш NO:233 CDR2 heavy zalifrelimab chains ISSSSSYI 8 SEQ Ш NO:234 CDR3 heavy chains zalifrelimab ARVGLMGPFD I 11 SEQIDNO:235 Zalifrelimab light chain CDR1 QSVSRY 6 SEQ Ш NO:236 Zalifrelimab light chain CDR2 GAS 3 SEQ Ш NO:237 Zalifrelimab light chain CDR3 QOYGSSPWT 9

Examples of additional anti-CTLA-4 antibodies include, but are not limited to: AGEN1181, BMS-986218, BCD-145, ONC-392, CS1002, REGN4659 and ADG116, which are known to those skilled in the art.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody described in any of the following patent publications: US 2019/0048096 A1; US 2020/0223907; US 2019/0201334; US 2019/0201334; US 2005/0201994; EP 1212422 B1; WO 2018/204760; WO 2018/204760; WO 2001/014424; WO 2004/035607; WO 2003/086459; WO 2012/120125; WO 2000/037504; WO 2009/100140; WO 2006/09649; WO2005092380; WO 2007/123737; WO 2006/029219; WO 2010/0979597; WO 2006/12168 and WO 1997020574, each of which is incorporated herein by reference. Additional antibodies to CTLA-4 are described in US patent Nos. 5811097, 5855887, 6051227 and 6984720; in PCT publications No. WO 01/14424 and WO 00/37504; and US Publication Nos. 2002/0039581 and 2002/086014; and/or US Patent Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281, each of which is incorporated herein by reference. In some embodiments, the anti-CTLA-4 antibody is, for example, those described in the following publications: WO 98/42752; US Patent Nos. 6,682,736 and 6,207,156; Hurwitz, et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 10067-10071 (1998); Camacho, et al., J. Clin. Oncol, 2004, 22, 145 (Abstract No. 2505 (2004) (antibody CP-675206); or Mokyr, et al., Cancer Res., 1998, 58, 5301-5304 (1998), each of which is incorporated herein document by reference.

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand as described in WO 1996/040915 (incorporated herein by reference).

In some embodiments, the CTLA-4 inhibitor is a nucleic acid inhibitor of CTLA-4 expression. For example, RNAi molecules to CTLA-4 may take the form of the molecules described in PCT Publication Nos. WO 1999/032619 and WO 2001/029058; US Publication Nos. 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, 2005/0100913, 2006/0024798, 2008/0050342, 2008/0081373, 2008/024 8576 and 2008/055443; and/or US Patent Nos. 6,506,559, 7,282,564, 7,538,095 and 7,560,438 (incorporated herein by reference). In some cases, RNAi molecules to CTLA-4 take the form of double-stranded RNAi molecules described in European Patent No. EP 1309726 (incorporated herein by reference). In some cases, RNAi molecules to CTLA-4 take the form of double-stranded RNAi molecules described in US Pat. Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In some embodiments, the CTLA-4 inhibitor is an aptamer described in International Patent Application Publication No. WO 2004/081021 (incorporated herein by reference).

In other embodiments, the anti-CTLA-4 RNAi molecules of the present invention are the RNA molecules described in US Pat. Nos. 5,898,031, 6,107,094, 7,432,249, and 7,432,250 and

- 142 045580 in European Application No. EP 0928290 (incorporated herein by reference).

In some embodiments, the present invention includes a method of treating a patient suffering from cancer, comprising the steps of administering a TIL regimen, the TIL regimen comprising a TIL product genetically modified to express a CCR, and further comprising the step of administering a CTLA-4 inhibitor. In some embodiments, the present invention includes a composition comprising: (i) a TIL product genetically modified to express a CCR, and (ii) a CTLA-4 inhibitor. In some embodiments, the present invention includes a kit comprising: (i) a TIL product genetically modified to express a CCR, and (ii) a CTLA-4 inhibitor.

In some embodiments, the present invention includes a method of treating a patient suffering from cancer, comprising the steps of administering a TIL regimen, the TIL regimen comprising a TIL product genetically modified to express a CCR, and further comprising the steps of administering a CTLA-4 inhibitor and either a PD-1 inhibitor, or a PD-L1 inhibitor. In some embodiments, the present invention includes a composition comprising: (i) a TIL product genetically modified to express a CCR, (ii) a CTLA-4 inhibitor, and (iii) either a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the present invention includes a kit comprising: (i) a TIL product genetically modified to express a CCR, (ii) a CTLA-4 inhibitor, and (iii) either a PD-1 inhibitor or a PD-L1 inhibitor.

4. Preconditioning of patients with lymphedema.

In one embodiment, the invention relates to a method of treating cancer using a population of TILs, in which the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of TILs in accordance with the present invention. In one embodiment, the invention includes a population of TILs for use in the treatment of cancer in a patient who has previously undergone non-myeloablative chemotherapy. In one embodiment, the TIL population is intended to be administered by infusion. In one embodiment, the nonmyeloablative chemotherapy is cyclophosphamide at a dose of 60 mg/kg/day for 2 days (days 27 and 26 before TIL infusion) and fludarabine at a dose of 25 mg/m ² /day for 5 days (days 27 to 23 before TIL infusion). In one embodiment, following non-myeloablative chemotherapy and TIL infusion (on day 0) in accordance with the present invention, the patient receives an intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) intravenously at a dose of 720,000 IU/kg every 8 hours until physiologically tolerated. In some embodiments, the TIL population is intended to treat cancer in combination with IL-2, wherein the IL-2 is administered after the TIL population.

Experimental evidence suggests that lymphodepletion prior to adoptive transfer of tumor-specific T cells plays a key role in increasing treatment efficacy by eliminating regulatory T cells and competing elements of the immune system (cytokine residues). Accordingly, in some embodiments, prior to administration of the TILs of the present invention, a lymphodepletion step (sometimes also referred to as immunosuppressive conditioning) is applied to the patient.

Typically, lymphodepletion is achieved by administering fludarabine or cyclophosphamide (the active form is called mafosfamide) and combinations thereof. Such methods are described in Gassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski, et al., Nat. Clin. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J. Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol. 2005, 23, 2346-2357, all of which are incorporated herein by reference in their entirety.

In some embodiments, fludarabine is administered at a concentration of 0.5 μg/ml to 10 μg/ml fludarabine. In some embodiments, fludarabine is administered at a concentration of 1 μg/mL fludarabine. In some embodiments, treatment with fludarabine is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or more. In some embodiments, fludarabine is administered at a dose of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day or 45 mg/kg/day. In some embodiments, treatment with fludarabine is administered for 2-7 days at a dose of 35 mg/kg/day. In some embodiments, treatment with fludarabine is administered for 4-5 days at a dose of 35 mg/kg/day. In some embodiments, treatment with fludarabine is administered for 4-5 days at a dose of 25 mg/kg/day.

In some embodiments, maffosfamide, the active form of cyclophosphamide, is obtained at a concentration of 0.5 μg/ml to 10 μg/ml by administering cyclophosphamide. In some embodiments, maffosfamide, the active form of cyclophosphamide, is produced at a concentration of 1 μg/ml by administering cyclophosphamide. In some embodiments, cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or more. In some embodiments, cyclophosphamide is administered at a dose of 100 mg/m ² /day, 150 mg/m ² /day, 175 mg/m ² /day, 200 mg/m ² /day, 225 mg/m ² /day, 250 mg /m ² /day, 275 mg/m ² /day or 300 mg/m ² /day. In some embodiments, cyclophosphamide is administered intravenously (i.e., IV). In some embodiments, treatment with cyclophosphamide is administered for 27 days at a dose of 35 mg/kg/day. In some embodiments, treatment with cyclophosphamide is administered for 4-5 days at a dose of 250 mg/m ² /day IV. In some embodiments, treatment with cyclophosphamide is administered for 4 days at a dose of 250 mg/m ² /day IV.

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In some embodiments, lymphodepletion is performed by co-administering fludarabine and cyclophosphamide to the patient. In some embodiments, fludarabine is administered at a dose of 25 mg/m ² /day IV and cyclophosphamide is administered at a dose of 250 mg/m ² /day IV for 4 days.

In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg/m ² /day for two days, followed by fludarabine at a dose of 25 mg/m ² /day for five days.

In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg/m ² /day for two days and administering fludarabine at a dose of 25 mg/m ² /day for five days, with both cyclophosphamide and fludarabine administered on the first two days, while lymphodepletion is carried out in just five days.

In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of about 50 mg/m ² /day for two days and administering fludarabine at a dose of about 25 mg/m ² /day for five days, both cyclophosphamide and fludarabine are administered. in the first two days, while lymphodepletion is carried out in just five days.

In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of about 50 mg/m ² /day for two days and administering fludarabine at a dose of about 20 mg/m ² /day for five days, both cyclophosphamide and fludarabine are administered. in the first two days, while lymphodepletion is carried out in just five days.

In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of about 40 mg/m ² /day for two days and administering fludarabine at a dose of about 20 mg/m ² /day for five days, both cyclophosphamide and fludarabine are administered. in the first two days, while lymphodepletion is carried out in just five days.

In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of about 40 mg/m ² /day for two days and administering fludarabine at a dose of about 15 mg/m ² /day for five days, both cyclophosphamide and fludarabine are administered. in the first two days, while lymphodepletion is carried out in just five days.

In one embodiment, lymphodepletion is performed by administering cyclophosphamide at a dose of 60 mg/ ^m2 /day and fludarabine at a dose of 25 mg/ ^m2 /day for two days, followed by fludarabine at a dose of 25 mg/ ^m2 /day for three days. .

In one embodiment, cyclophosphamide is administered with mesna. In one embodiment, mesna is administered at a dose of 15 mg/kg. In the embodiment in which mesna is infused, and when infused continuously, mesna can be administered over approximately 2 hours with cyclophosphamide (on days -5 and/or -4) and then at a rate of 3 mg/kg/hour for the remaining 22 hours for 24 hours, starting simultaneously with each dose of cyclophosphamide.

In one embodiment, lymphodepletion includes the step of treating a patient with an IL-2 regimen, starting the day after administration of the third population of TILs to the patient.

In one embodiment, lymphodepletion includes the step of treating the patient with an IL-2 regimen, starting on the same day that the third population of TILs is administered to the patient.

In some embodiments, lymphodepletion includes 5 days of preconditioning. In some embodiments, the days are indicated as days -5 to -1 or day 0 to day 4. In some embodiments, the regimen includes cyclophosphamide on days -5 and -4 (ie, days 0 and 1). In some embodiments, the regimen includes intravenous administration of cyclophosphamide on days -5 and -4 (ie, days 0 and 1). In some embodiments, the regimen includes intravenous administration of cyclophosphamide at a dose of 60 mg/kg on days -5 and -4 (ie, days 0 and 1). In some embodiments, cyclophosphamide is administered with mesna. In some embodiments, the regimen further includes fludarabine. In some embodiments, the regimen further includes intravenous administration of fludarabine. In some embodiments, the regimen further includes intravenous administration of fludarabine at a dose of 25 mg/m ² . In some embodiments, the regimen further includes intravenous administration of fludarabine at a dose of 25 mg/m ² on days -5 and -1 (ie, days 0 to 4). In some embodiments, the regimen further includes intravenous administration of fludarabine at a dose of 25 mg/m ² on days -5 and -1 (ie, days 0 to 4).

In some embodiments, the nonmyeloablative lymphodepletion regimen includes the steps of administering cyclophosphamide at a dose of 60 mg/m ² /day and fludarabine at a dose of 25 mg/m ² /day for two days, followed by administration of fludarabine at a dose of 25 mg/m ² /day for five days.

In some embodiments, the nonmyeloablative lymphodepletion regimen includes the steps of administering cyclophosphamide at a dose of 60 mg/m ² /day and fludarabine at a dose of 25 mg/m ² /day for two days, followed by administration of fludarabine at a dose of 25 mg/m ² /day for three days.

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. 26.

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Table 26

Illustrative diagram. lymphodepletion and treatment.

Day -5 -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 60 mg/kg X X Mesna (if necessary) X X Fludarabine 25 mg/ ^m2 /day X X X X X TIL infusion X

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. 27.

Table 27

Illustrative scheme of lymphodepletion and treatment__________________

Day -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 60 mg/kg X X Mesna (if necessary) X X Fludarabine 25 mg/ ^m2 /day X X X X TIL infusion X

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. 28.

Table 28

Illustrative scheme of lymphodepletion and treatment _____________

Day -3 -2 -1 0 1 2 3 4 Cyclophosphamide 60 mg/kg X X Mesna (if necessary) X X Fludarabine 25 mg/ ^m2 /day X X X TIL infusion X

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. 29.

Table 29 ___________________Illustrative diagram of lymphodepletion and treatment, ____

Day -5 -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 60 mg/kg X X Mesna (if necessary) X X Fludarabine 25 mg/ ^m2 /day X X X Day -5 -4 -3 -2 -1 0 1 2 3 4 TIL infusion X

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. thirty.

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Table 30

Illustrative diagram of lymphodepletion and treatment

Day -5 -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 300 mg/kg X X Mesna (if necessary) X X Fludarabine 30 mg/ ^m2 /day X X X X X TIL infusion X

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. 31.

Table 31

Illustrative diagram of lymphodepletion and treatment

Day -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 300 mg/kg X X Mesna (if necessary) X X Fludarabine 30 mg/ ^m2 /day X X X X TIL infusion X

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. 32.

Table 32

Illustrative diagram of lymphodepletion and treatment

Day -3 -2 -1 0 1 2 3 4 Cyclophosphamide 300 mg/kg X X Mesna (if necessary) X X Fludarabine 30 mg/ ^m2 /day X X X TIL infusion X

In some embodiments, a non-myeloablative lymphodepletion regimen is used in accordance with Table. 33.

Table 33

Illustrative scheme of lymphodepletion and treatment___________________

Day -5 -4 -3 -2 -1 0 1 2 3 4 Cyclophosphamide 300 mg/kg X X Mesna (if necessary) X X Day -5 -4 -3 -2 -1 0 1 2 3 4 Fludarabine 30 mg/ ^m2 /day X X X TIL infusion X

In some embodiments, the TIL infusion used with the above embodiments of myeloablative lymphodepletion regimens may be any TIL composition described herein, including TIL products genetically modified to express CCR as described herein, and may also include MIL infusions and PBL instead of TIL infusion, as well as the addition of IL-2 regimens and administration of concomitant medications (eg, PD-1 and PD-L1 inhibitors) as described herein.

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5. IL-2 patterns.

In one embodiment, the IL-2 regimen comprises a high-dose IL-2 regimen, wherein the high-dose IL-2 regimen comprises aldesleukin or a biosimilar or variant thereof administered intravenously beginning on the day after administration of a therapeutically effective portion of the TIL therapeutic population, wherein aldesleukin or a biosimilar or variant thereof is administered at a dose of 0.037 mg/kg or 0.044 mg/kg IU/kg (patient body weight) via 15-minute intravenous bolus infusions every eight hours until tolerated, for a maximum of 14 doses. After 9 days of rest, this schedule can be repeated for 14 more doses, for a total of 28 doses maximum. In some embodiments, IL-2 is administered in 1, 2, 3, 4, 5, or 6 doses. In some embodiments, IL-2 is administered at a maximum dose of up to 6 doses.

In one embodiment, the IL-2 regimen includes a decrescendo IL-2 regimen. Decrescendo IL-2 regimens are described in O'Day, et al., J. Clin. Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, the description of which is incorporated herein by reference. In one embodiment, the IL-2 decrescendo regimen includes IV administration of 18x106 IU/ ^m2 aldesleukin or a biosimilar or variant thereof over 6 hours, followed by IV administration of 18x106 IU/ ^m2 over 12 hours, followed by IV administration of 18x106 IU/m2. m ² for 24 hours, followed by intravenous administration of 4.5x106 IU/m ² for 72 hours. This treatment cycle can be repeated every 28 days, for a maximum of four cycles. In one embodiment, the decrescendo IL-2 regimen includes 18,000,000 IU/ ^m2 on day 1, 9,000,000 IU/ ^m2 on day 2, and 4,500,000 IU/ ^m2 on days 3 and 4.

In one embodiment, the IL-2 regimen includes a low dose IL-2 regimen. Any low dose IL-2 regimen known in the art can be used, including the low dose IL-2 regimens described in Dominguez-Villar and Hafler, Nat. Immunology 2000, 19, 665673; Hartemann, et al., Lancet Diabetes Endocrinol. 2013, 1, 295-305; and Rosenzwaig, et al., Ann. Rheum. Dis. 2019, 78, 209-217, the description of which is incorporated herein by reference. In one embodiment, a low-dose IL-2 regimen includes 18x106 IU per ^m2 of aldesleukin or a biosimilar or variant thereof over 24 hours, administered as a continuous infusion for 5 days, followed by 2-6 days without IL-2 therapy, optionally followed by an additional 5 days of intravenous administration of aldesleukin or its biosimilar or variant as a continuous infusion of 18x106 IU per m ² over 24 hours, optionally followed by 3 weeks without IL-2 therapy, after which additional cycles may be administered.

In one embodiment, the IL-2 regimen includes administering pegylated IL-2 every 1, 2, 4, 6, 7, 14, or 21 days at a dose of 0.10 mg/day to 50 mg/day. In one embodiment, the IL2 regimen includes administering bempegaldesleukin or a fragment, variant, or biosimilar thereof every 1, 2, 4, 6, 7, 14, or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In one embodiment, the IL-2 regimen includes administration of THOR-707 or a fragment, variant, or biosimilar thereof every 1, 2, 4, 6, 7, 14, or 21 days at a dose of 0.10 mg/day up to 50 mg/day.

In one embodiment, the IL-2 regimen includes administration of nemvaleukin alfa or a fragment, variant, or biosimilar thereof every 1, 2, 4, 6, 7, 14, or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In one embodiment, the IL-2 regimen includes administering an IL-2 fragment grafted to an antibody scaffold. In one embodiment, the IL-2 regimen involves administering an antibody-cytokine-coupled protein that binds to the low-affinity IL-2 receptor. In one embodiment, the antibody-cytokine grafted protein comprises a heavy chain variable region (VH) including the complementarity determining regions HCDR1, HCDR2, HCDR3; light chain variable region (VL), including LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into CDR V _H or VL, wherein the antibody-cytokine grafted protein preferentially expands effector T cells over regulatory T cells. In one embodiment, the antibody-cytokine grafted protein comprises a heavy chain variable region ( _VH ) including the complementarity determining regions HCDR1, HCDR2, HCDR3; light chain variable region (VL), including LCDR1, LCDR2, LCDR3; and an IL-2 molecule or fragment thereof grafted into a VH or VL CDR, wherein the IL-2 molecule is a mutein, and wherein the antibody-cytokine grafted protein preferentially expands effector T cells over regulatory T cells. In one embodiment, the IL-2 regimen includes administering an antibody comprising a heavy chain selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 38, and a light chain selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 39, or a fragment, variant or biosimilar thereof, every 1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50 mg/day.

In some embodiments, the antibody-cytokine grafted protein described herein has a longer serum half-life than a wild-type IL-2 molecule, such as, but not limited to, aldesleukin (Proleukin®) or a comparable molecule.

In some embodiments, the TIL infusion used with the above embodiments of myeloablative lymphodepletion regimens can be any composition

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TIL described herein and may also include infusions of MIL and PBL instead of infusion

TIL, as well as the addition of IL-2 regimens and administration of concomitant medications (eg, PD-1 and PD-L1 inhibitors) as described herein.

In some embodiments, the present invention includes a method of treating a patient suffering from cancer, comprising the step of administering a TIL regimen, the TIL regimen comprising a TIL product genetically modified to express a CCR, and further comprising the step of administering an IL-2 regimen. In some embodiments, the present invention includes a composition comprising: (i) a TIL product genetically modified to express a CCR, and (ii) an IL-2 regimen. In some embodiments, the present invention includes a kit comprising: (i) a TIL product genetically modified to express a CCR, and (ii) an IL-2 regimen.

In some embodiments, the present invention includes a method of treating a patient suffering from cancer, comprising the steps of administering a TIL regimen, the TIL regimen comprising a TIL product genetically modified to express a CCR, and further comprising the steps of administering an IL-2 regimen and administering either a PD-1 inhibitor , or a PD-L1 inhibitor. In some embodiments, the present invention includes a composition comprising: (i) a TIL product genetically modified to express a CCR, (ii) an IL-2 regimen, and (iii) either a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the present invention includes a kit comprising: (i) a TIL product genetically modified to express a CCR, (ii) an IL-2 regimen, and (iii) either a PD-1 inhibitor or a PD-L1 inhibitor.

In some embodiments, the present invention includes a method of treating a patient suffering from cancer, comprising the steps of administering a TIL regimen, the TIL regimen comprising a TIL product genetically modified to express a CCR, and further comprising the step of administering a CTLA-4 inhibitor and administering an IL-2 regimen. In some embodiments, the present invention includes a composition comprising: (i) a TIL product genetically modified to express a CCR, (ii) a CTLA-4 inhibitor, and (iii) an IL-2 regimen. In some embodiments, the present invention includes a kit comprising: (i) a TIL product genetically modified to express a CCR, (ii) a CTLA-4 inhibitor, and (iii) an IL-2 regimen.

In some embodiments, the present invention includes a method of treating a patient suffering from cancer, comprising the steps of administering a TIL regimen, the TIL regimen comprising a TIL product genetically modified to express a CCR, and further comprising the steps of administering an IL-2 regimen, administering a CTLA-4 inhibitor, and either a PD-1 inhibitor or a PD-L1 inhibitor. In some embodiments, the present invention includes a composition comprising: (i) a TIL product genetically modified to express a CCR, (ii) an IL-2 regimen, (iii) either a PD1 inhibitor or a PD-L1 inhibitor, and (iv) a CTLA-2 inhibitor. 4. In some embodiments, the present invention includes a kit comprising: (i) a TIL product genetically modified to express a CCR, (ii) an IL-2 regimen, (iii) either a PD-1 inhibitor or a PD-L1 inhibitor, and (iv) an inhibitor CTLA-4.

B. Adoptive cell transfer.

Adoptive cell transfer (ACT) is an effective form of immunotherapy and involves the transfer of immune cells with antitumor activity into the body of patients suffering from cancer. ACT is a treatment approach that involves in vitro identification of lymphocytes with antitumor activity, in vitro expansion of these cells to large numbers of cells, and infusion of them into a host suffering from cancer. Lymphocytes used for adoptive transfer can be obtained from the stroma of resected tumors (tumor infiltrating lymphocytes or TILs). TIL for ACT can be obtained as described herein. In some embodiments, TILs are prepared, for example, in accordance with the method described in FIG. 2A and/or 9. They may also be derived from, or originate from, blood if they are genetically modified to express antitumor T-cell receptors (TCRs) or chimeric antigen receptors (CARs), enriched in mixed tumor lymphocyte cultures (MLTC), or cloned using autologous antigen-presenting cells and tumor-derived peptides. ACT in which lymphocytes are obtained from a cancer-bearing host to whom they will be reinfused is called autologous ACT. US Patent Publication No. 2011/0052530 describes a method of performing adoptive cell therapy to promote cancer regression, primarily in the treatment of patients suffering from metastatic melanoma, the full description of these methods is incorporated by reference. In some embodiments, TILs may be administered as described herein. In some embodiments, TILs may be administered as a single dose. Such administration may be by injection, for example intravenous injection. In some embodiments, TILs and/or cytotoxic lymphocytes may be administered in multiple doses. Dosing can be done once, twice, three times, four times, five times, six times or more than six times a year. Dosing can be done once a month, once every two weeks, once a week, or once every other day. Administration of TILs and/or cytotoxic lymphocytes can be continued for as long as necessary.

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V. Coordination of cell proliferation product production and patient treatment events.

As described herein, the time from tumor resection in a patient to completion of TIL production varies depending on several factors, including, for example, the size of the tumor obtained from the patient, the number of cells at the end of the various production steps, the number of days during which the various production stages, etc. As a result, some flexibility is required when planning a patient's various treatment events. In some embodiments, the patient's treatment event includes lymphodepletion. In some embodiments, the patient's treatment event includes an infusion of TIL. In some embodiments, the patient's treatment event includes a leukapheresis procedure. In some embodiments, the patient's treatment event involves administration of an IL-2 regimen. In some embodiments, the patient's treatment event includes tumor resection. In some embodiments, the patient's treatment event includes a hospital stay for treatment following a procedure.

An aspect of the present invention provides a system for implementing the methods described herein. The system may include a patient portal, a manufacturing portal, a physician portal, and a logistics portal. The Patient Portal allows a patient or a person related to a patient (e.g., caregiver, guardian, or legal representative) (collectively referred to herein as the patient for convenience) to access information related to the patient's schedule of treatment events. The patient portal may require the patient to provide authentication information to access this information. The patient portal may also allow the patient to change information related to the patient, such as, for example, address or other personal information.

The physician portal, also called the hospital-side interface, allows the clinic (i.e., clinic staff) to access and/or change information related to the patient, the manufacturing process, the manufacturing facility, the logistics provider, and information related to various patient treatment events that are or will be performed in the clinic.

The production portal communicates with the hospital-side interface and the logistics portal/logistics interface to determine and transmit information related to the production process and/or availability of production slots. The production portal also allows you to create production labels containing updated quality information at various stages of production, as described in this document.

The logistics portal, also referred to herein as the logistics interface, communicates with the manufacturing portal, and the hospital-side interface exchanges information related to the delivery schedule of the material (i.e., solid tumor or manufactured cell therapy product) between the clinic and the manufacturing facility, including, for example, production scheduling, availability of production slots, and scheduling of patient treatment events to facilitate timely delivery of material.

A system for implementing the various methods described herein may be implemented on a computer system either by implementing a proprietary computer program or by appropriately modifying a commercially available software platform such as, for example, those provided by Vineti, Salesforce.com, Salesforce Health Cloud, IQVIA , TracSel and SAP.

Modification of a commercially available software platform to properly implement the system and perform one or more of the methods described herein may enable the platform to perform processes for which it was not originally intended. In other words, modification in some embodiments may be based on non-traditional use of various tools provided by the platform. In some embodiments, a system for implementing the various methods described herein may include a software platform, such as a framework, that integrates an enterprise resource planning (ERP) system to automate logistics tasks and a manufacturing execution system (MES) providing automation of production tasks. An example of such a framework is described in US Pat. No. 7,343,605, which is incorporated herein by reference in its entirety.

For example, in addition to MES applications, applications from the enterprise management layer (enterprise resource planning layer) and automation layer (control layer) can also be integrated via a framework and monitored or controlled via a workstation (for example, in a hospital, manufacturing plant or courier service). In this way, the framework can become an integration platform for the entire patient treatment process, including patient registration, tumor acquisition, tumor delivery to the manufacturing facility, production of the therapeutic or expanded cell therapy product, delivery of the cell therapy therapeutic product to the treatment facility, administration of the therapy to the patient, and beyond. patient treatment. Various applications of the plant control level, MES level and automation level can be integrated easily and cost-effectively by the framework using adapters and/or shells. Therefore, the framework should be considered as a middleware platform and as a tool for integrating production applications. The end user (eg, curator) can see the relevant status of applications to be monitored through the workstation, and can also access application data and methods. The end user can then connect applications to each other using this access.

Thus, the framework allows, firstly, to achieve vertical integration of applications from different levels of the enterprise, and secondly, the framework provides horizontal integration of applications at the MES level.

For example, a card software object that allows information to be stored on a platform is a general definition of the type of information stored. For example, a card software object on a ready-made platform allows you to store information about customer requests. For such an object, there may be multiple records that store information about specific instances of that data type. For example, a card entry for storing information about a person's training request and another card entry for storing information about another person's configuration problem. These objects can be configured to store information related to, for example, a patient, the patient's treatment events for that patient, and the associated schedule.

Likewise, automated actions, such as notifications and alerts, and workflow rules, such as business logic actions provided in the out-of-the-box platform, can be modified to trigger changes in schedules and process steps, such as changes in production processes and subsequent changes in production schedules, production slot availability, and patient treatment event schedules based on quality assurance (QA) results.

In some embodiments, the system described herein provides quality assurance functions for identifying likely points of failure during a manufacturing process, such as those described in US Patent No. 10747209 and US Patent No. 7799273, which are incorporated herein by reference in their entirety.

In some embodiments, the system described herein provides data systems configured to maintain the integrity of electronic batch records created during the production of a cell therapy end product from a solid tumor (or fragment thereof) obtained from a patient, such as those described in US Pat. No. 8,491,839, which is incorporated herein by reference in its entirety. In some embodiments, the system described herein includes application software adapted for use in a cell production process, wherein the cell production process produces an expanded cell therapy product, such as a therapeutic population of T cells. The application software is configured to control a plurality of devices included in a closed cell production system, such as gas permeation devices.

In some embodiments, the system described herein integrates the application software and cell production methods described herein to provide a comprehensive testing and quality assurance protocol that is used by multiple end users, wherein data collected from the system is analyzed and are used to determine whether quality assurance protocols and inspection protocols are being followed.

In some embodiments, the system applies application software to multiple production lines and/or multiple cell production facilities, while monitoring and controlling multiple cell production lines and multiple production lines using the same system.

In some embodiments, the system implements the methods described herein in batch optimization of cell culture systems of a cell production process, wherein data collected by the cell culture system is continuously monitored over time and the data is used to analyze the cell culture system. In addition, this data is integrated and used to analyze the quality control of the cell production process as a whole.

In some embodiments, the system described herein allows workstations to be designed to perform various steps in the manufacturing process for the final cell therapy product. Workstations may be configured to examine data associated with a previous step to ensure the integrity of the process being performed and, where possible, to take corrective action. Examples of such workstation designs are presented in US Pat. No. 8,041,444, which is incorporated herein by reference in its entirety.

In some embodiments, the system described herein facilitates coordination between a hospital facility and various manufacturers capable of producing a cell therapy product from a solid tumor obtained from the hospital facility by providing access to available production schedules from various manufacturers.

Examples of systems providing such coordination are presented in US Pat. No. 8,069,071, which is incorporated herein by reference in its entirety.

In some embodiments, the system described herein is based on a cloud-based architecture that allows various organizations involved in the production of a cell therapy product to gain real-time access (with appropriate permissions) to information related to the production and transportation of the cell therapy product . Examples of such cloud architecture are presented in US Patent No. 9965562, which is incorporated herein by reference in its entirety.

In some embodiments, the system described herein allows personnel associated with obtaining a tumor from a patient, manufacturing a cell therapy product from the resulting tumor, and transporting the tumor and cell therapy product to identify and authenticate themselves using electronic signatures to manage and approve processes. , as described in US Patent Application Publication No. 2004/0243260, which is incorporated herein by reference in its entirety.

In some embodiments, the system may further comprise or include a customer relationship management (CRM) subsystem.

The CRM subsystem can archive information related to various parties, such as, for example, doctors, hospital sites, nurses, billers, business contacts, etc. The information is maintained with the CRM engine to ensure that a single view of the parties is maintained in one unified sequence, allowing the views of prospects and parties on the platform to be shared in a single sequence in a confidential manner within the system. Information related to the relationship between the parties can be shared in a distributed context in a master-master, slave-slave, or peer-to-peer mode, partially or completely anonymously by removing personal information from one or more distributed slave or peer CRM systems. One example of such a CRM subsystem is described in US Patent Publication No. 2014/0244351, US Patent No. 8489451 and US Patent No. 9342292, each of which is incorporated herein by reference in its entirety.

In some embodiments, the system described herein may be configured to provide a network that includes a central event processing subsystem for receiving, processing, and routing messages caused by real-time medical events and/or industrial events. For example, a central event processing system may identify patient information associated with a medical event and match the patient information with one or more of a health plan, clinic location, TIL manufacturing facility, and/or logistics provider. After associating a medical and/or occupational event with stakeholders, the central event processing subsystem may forward at least a portion of the information related to the medical and/or occupational event to one or more interested parties within a short period of time after the triggering event (e.g., substantially real time). For example, based on the rules associated with each stakeholder, the central event processing system may forward information and/or issue an alert or notification to the stakeholder so that the stakeholder is aware of the medical event. An example of such a central event processing subsystem is described in US Patent Publication No. 2014/0372147 and US Patent No. 10242060, each of which is incorporated herein by reference in its entirety.

In some embodiments, the system described herein is configured to select a workflow for a cancer treatment regimen, including patient treatment events, such as, for example, tumor resection, lymphodepletion, leukapheresis, TIL infusion and/or IL-2 regimen, or others patient treatment events described herein. Once the workflow is selected, the system may create a purchase order corresponding to the cell infusion therapy on behalf of the patient, wherein the order corresponding to the cell infusion therapy includes at least one of a request for ordering cells and a request for a specified treatment regimen corresponding to the cancer treatment. Examples of such a workflow selection subsystem are described in US Patent Publication No. US 2014/0088985, which is incorporated herein by reference in its entirety.

In some embodiments, the system may include a telephony subsystem, which may include authenticating requests for service, including authenticating a remote access device prior to text or audio communication with the patient or patient representative. In some embodiments, authentication may be accomplished by automatically authenticating the remote access device or asking questions of the patient (or representative). An example of such a telephony subsystem and authentication method is described in the patent publication

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US No. US 2019/0026747, which is incorporated herein by reference in its entirety.

A. System for coordinating the production of cell reproduction product.

Embodiments of the present invention include a method and system for coordinating the production of a cell therapy product, such as, for example, T cells or TILs for a patient, and dynamically scheduling various stages of the manufacturing process, as well as various patient treatment events based on the progress and success of the various stages production process.

The methods and systems described herein are further configured to provide a specific technical advantage over existing systems for providing a continuous and automatic chain of custody and chain of identification for a patient-specific biological sample during an immunotherapy procedure to create a computerized information portal that interested parties, such as the patient, physician, manufacturer and other healthcare personnel, can be used to quickly understand and track the current phase of an immunotherapy procedure and the status of the patient's biological sample during the procedure. Lack of ability to maintain a chain of responsibility and chain of identification, resulting in delays during the production process that can have immeasurable consequences for a patient suffering from a life-threatening illness.

Embodiments of the present invention provide for maintaining a chain of custody for biological material during the manufacturing process (including quality assurance, production, finished product inspection, and completion of shipment), as well as a chain of responsibility for biological material during the delivery process of the final product (including shipping and on-site delivery). infusion). Embodiments of the present invention further enable a chain of custody to be continuously and permanently associated with a specific patient, thereby providing a complete chain of identification between the patient and the biological material throughout all stages of production.

Maintaining the Chain of Responsibility (COC) and Chain of Identity (COI) is accomplished by linking each event along the biological material's journey from the patient through the transport, manufacturing process, and transport back to the patient with a cell order ID and a patient-specific identifier (i.e., unique for the patient) and tracking each event along the way.

In addition, the methods and systems described herein are designed to integrate and synchronize the acquisition of living tissue from a patient with the manufacturing process, and to enable verification of each step from the acquisition of living tissue to the administration of the cell therapy product and subsequent treatment events for SOS and COI.

The methods and systems described herein are further configured to coordinate logistics for obtaining living tissue from a patient, delivering living tissue to a selected manufacturing facility, manufacturing a cell therapy product at a selected manufacturing facility, and delivering the cell therapy product from the manufacturing facility to a clinic for treatment. patient.

In fig. 3A is a block diagram of a system for coordinating the production of a cell therapy product for a patient in accordance with an embodiment of the present invention. In some embodiments, the functions performed by the components in FIG. 3A can be integrated into one component. Additionally, in some embodiments, the functions performed by one or more components in FIG. 3A may also be implemented by other components in FIG. 3A.

As shown in FIG. 3A, system 300 may include a hospital-side interface 110, an event scheduler 120, a logistics interface (also referred to herein as a courier portal) 130, and/or a manufacturing portal 140. Each of the hospital-side interface 110, an event scheduler 120, a logistics interface 130 and production portal 140 communicates with the other three, for example, using a communications network such as a LAN, WAN (eg, the Internet), and/or a cellular network.

In some embodiments, the hospital-side interface 110, event scheduler 120, logistics interface 130, manufacturing portal 140, and related modules (shown, for example, in FIG. 3) are suitably modified or built on commercially available software platforms, such as, for example, provided by Vineti, Salesforce.com, Salesforce Health Cloud, IQVIA, TracSel and SAP.

The hospital-side interface 110 may be associated with or interact with a hospital or other agency responsible for administering a patient's treatment, such as providing cancer therapy, as described herein. In some embodiments, the hospital-side interface 110 is associated with a clinical facility that performs the procedure of obtaining a solid tumor from a patient and obtaining a cell therapy product from the solid tumor or fragments thereof. In some embodiments, the clinical site operates only to obtain a solid tumor, while the process of obtaining a cell therapy product from a solid tumor or fragments thereof may be carried out in a manufacturing facility.

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In some embodiments, the hospital-side interface 110 is associated with the hospital that infuses the expanded cell therapy product obtained from the manufacturing facility and provides follow-up care and/or any prior or subsequent treatment to the patient. Clinicians or employees of a hospital or clinic may interact with the system 300 through a hospital-side interface 110 .

In some embodiments, hospital-side interface 110 includes one or more computing devices, such as, for example, a desktop computer, a cloud server, a laptop computer, mobile devices, or any other computing device, including hardware and software modules that execute on a processor. and interact with the storage device. The hospital-side interface 110 may be connected via a wired or wireless connection to computing devices operated in or associated with the hospital or clinical facility.

In some embodiments, the hospital-side interface 110 may include a tracking module 112 configured to track biological material (e.g., a patient's tumor, T cells obtained from a patient, expanded T cells at various stages of the T cell expansion process, etc.). d.) from the patient from the moment of removal from the patient’s body until the moment of infusion into the patient’s body.

In some embodiments, when a patient registers for a TIL infusion treatment, a cell order request is created to produce an expanded cell therapy product for the patient and a cell order ID associated with the cell order request is generated. In some embodiments, a purchase order is generated pursuant to a cell order request and loaded into the system from the hospital facility. In some embodiments, the system may include an interface for generating and/or downloading a purchase order.

In addition, a patient-specific ID is generated, unique to the patient, which is linked to the cell order ID. In some embodiments, the patient-specific identifier may include, for example, a patient identifier with a sequential suffix, where the suffix is a single letter added sequentially for each patient (A-Z). The patient-specific identifier is unique in the system. In some embodiments, a patient-specific identifier may be visible only to certain individuals (described in detail elsewhere herein). In some embodiments, a patient-specific identifier is printed on every label printed throughout the journey of the patient's biological material, as well as on the manufactured biological material. In some embodiments, the patient-specific identifier is read-only by all users of the system. In other words, the patient-specific identifier in some embodiments cannot be edited once it is generated.

In some embodiments, the cell order identifier may include information such as, for example, a unique patient identifier, a cell order identifier, an order code, a cell order lot number, values of one or more acceptance parameters at various points in time, one or more indicators, indicating whether acceptance criteria are met at different points in time, or a combination of these.

In some embodiments, the cell order identifier and the patient-specific identifier are scanned at each time that any biological material (e.g., a solid tumor removed from a patient, fragments thereof, a cell therapy product removed therefrom, or an expanded cell therapy product, obtained by propagating a cell therapy product) associated with a patient, transferred or processed. Each scan can be recorded and verified by the tracking module 112 throughout the entire process from the time it is removed from the patient to the time it is infused into the patient. Validating the patient-specific ID at every step throughout the process ensures the product's Chain of Identity (COI), while validating the cell order ID at every step throughout the process ensures the product's Chain of Responsibility (COI). Detailed information related to maintaining COI and SOS is provided elsewhere in this document.

In some embodiments, the scanned information is verified against a purchase order generated by the hospital facility. For example, when a delivery is accepted at a manufacturing facility, the label on the shipping container can be scanned and the information on the label can be checked against the purchase order to ensure accuracy. In some embodiments, the result of the test is recorded in the system.

It should be understood that although tracking module 112 is shown in FIG. 3A and described herein as part of the hospital-side interface 110, the tracking module 112 may be implemented as a stand-alone computing device having a processor and storage device in some embodiments. Likewise, tracking module 112 may also be implemented as a stand-alone software module (eg, stored on a cloud server) in some embodiments.

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In some embodiments, the hospital-side interface 110 may provide limited access to the patient through the patient access module 116 to allow the patient to obtain information related to treatment procedures and related schedules. The patient access module 116 may also allow the patient to provide information related to himself, such as, for example, personal identification information, insurance information, and information related to his medical condition, to clinicians.

The hospital-side interface 110 may further include a retrieval module 114 that allows clinical facility personnel to obtain a solid tumor from a patient according to a predetermined protocol and enter information related to the procedure for obtaining a solid tumor from a patient. Information related to a solid tumor procedure may, in some embodiments, be archived to allow for post-facto auditing to ensure compliance with regulatory requirements.

In some embodiments, acquisition module 114 may further allow clinical facility personnel to provide the manufacturing facility with information about the solid tumor and the processes used to obtain the solid tumor from the patient. In embodiments in which the clinical site may also obtain a cell therapy product from a solid tumor, acquisition module 114 may allow clinical site personnel to provide information about the cell therapy product received, such as, for example, the process or procedure used to obtain the cell therapy product. and the results of quality control analyzes performed on the resulting cell therapy product by the manufacturing facility.

In some embodiments, the hospital-side interface 110 may further include a production coordination module 118 . The production coordination module 118 is configured to provide users at a clinical site with information related to one or more production sites, such as, for example, the availability of production slots and capabilities available at each of the one or more production sites. The manufacturing coordination module 118 further allows users in a clinical setting to coordinate the reservation of a manufacturing slot at a selected manufacturing facility and coordinate with a logistics provider through the logistics interface 130 to arrange for the transportation of a solid tumor, tumor fragments, or cell therapy product obtained from a patient. The manufacturing coordination module 118 further allows users in a clinical setting to obtain information regarding the manufacturing process and/or quality control analyzes performed during the manufacturing process through the manufacturing portal 140 .

The event scheduler 120 is configured to coordinate the scheduling of various activities performed in a clinical setting and a manufacturing plant. Additionally or alternatively, the event scheduler 120 may provide information related to scheduling various activities to a logistics service provider to facilitate transport of a cell therapy product between a clinical facility and a manufacturing facility, a facility where the cell therapy product is manufactured, such as a facility where the product is multiplied. cell therapy. As described in more detail below, based on various events that occur during the production of a cell therapy product, the event scheduler is configured to dynamically schedule when other event(s) should occur.

In some embodiments, the event scheduler 120 includes a computing device 122, such as, for example, a desktop computer, a server, a laptop computer, mobile devices, or any other computing device, including hardware and software modules that execute on a processor and interact with a storage device. . The hardware and/or software modules include an eligibility determination module 123 and a scheduling module 125. In some embodiments, the computing device 122 may be a cloud server accessible through the hospital interface 110, the manufacturing portal 140, and the logistics interface 130. The event scheduler 120 may be connected via a wired or wireless connection to computing devices operated within the manufacturing plant and/or hospital facility or associated with them.

It should be understood that although the acceptance determination module 123 is shown in FIG. 3A, as part of the event scheduler 120, the acceptance determination module 123 may also be implemented as part of the production portal 140. Alternatively, the acceptance determination module 123 may also be implemented as a stand-alone software module, for example, hosted on a cloud server.

The logistics interface or courier portal 130 may be associated with or interact with a logistics service provider, such as, for example, a courier or parcel service.

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In some embodiments, the logistics interface 130 includes one or more computing devices, such as, for example, a desktop computer, a cloud server, a laptop computer, mobile devices, or any other computing device, including hardware and software modules that execute on a processor and communicate with each other. with a storage device. Logistics interface 130 may be connected via a wired or wireless connection over a network, such as the Internet, to computing devices operated by or associated with a logistics service provider.

The manufacturing portal 140 may be associated with a manufacturing facility that produces an expanded cell therapy product. The production portal 140 allows personnel at a manufacturing facility to monitor and record various processes during the production of an expanded cell product, including, for example, maintaining a chain of identity and chain of responsibility, recording information related to quality control analyzes conducted during production, recording transitions between various processes and providing labels for cell therapy product containers during the propagation process.

In some embodiments, the manufacturing portal 140 includes one or more computing devices, such as, for example, a desktop computer, a cloud server, a laptop computer, mobile devices, or any other computing device, including hardware and software modules that execute on a processor and communicate with each other. with a storage device. The manufacturing portal 140 may be connected via a wired or wireless connection to computing devices operated in or associated with the manufacturing plant.

In some embodiments, production portal 140 includes a labeling module 142 and a COC/COI module 144 . The labeling module 142 allows manufacturing plant personnel to generate labels for containers containing a cell therapy product during the production of an expanded cell therapy product (also referred to herein as an expansion process). Labels may include information such as, for example, a patient-specific identifier, an identifier related to personnel handling the container and/or performing the current and/or previous step in the propagation process, the results of quality control passed in the previous step, a reason code related to to the reason for generating the label, bar code or two-dimensional code (eg, QR code) identifying the cell therapy product with a patient-specific identifier, and other appropriate information.

The COC/COI module 144 allows users associated with a production facility to maintain the ability to audit the chain of custody during the propagation process. The COC/COI module 144 further allows users associated with a manufacturing facility to maintain the ability to audit the chain of patient identification associated with an expanded cell therapy product.

In fig. 3B shows an object diagram for components of system 300 that are suitably modified or built upon commercially available software platforms in addition to standard components for those platforms. For example, a commercially available software platform may have built-in objects corresponding to patient access (including, for example, patient ID, patient contact information, patient authentication information, etc.), treatment plan (including, for example, patient initial treatment event schedule), clinician access (eg, clinician ID and clinician authentication information) and manufacturing access (eg, contact information for manufacturing facilities).

On the other hand, objects such as tumor acquisition forms associated with the procedure for obtaining a solid tumor from a patient, a production order including information related to the processes that will be used to produce the expanded cell therapy product, and information related to how the solid tumor has been processed, a production schedule at one or more manufacturing plants, a label audit trail to provide an audit of the entire process, may be specifically created to interact with embedded objects to provide specific functionality associated with the system 300.

Special objects and built-in objects are configured in the system 300 to interact with each other to provide support and control of SOS and COI throughout events from the receipt of a solid tumor from a patient to the infusion of an expanded cell therapy product into the patient. Dedicated facilities combined with built-in facilities, and planning of patient treatment events and associated logistics based on the progress of manufacturing processes.

For example, in some embodiments, special objects such as batch records and label audit trails may be provided in the system 300. These special objects, in addition to special workflow automation tools and special user profiles, provide a commercially available software platform with functions such as COC/COI support, auditability, dynamic treatment scheduling

- 155 045580 production management-based patient ties, production management-based label generation, and/or interactions between production management and receiving that were not originally provided for in a commercially available software platform. Additional information about the interaction between custom objects and built-in objects for the tumor acquisition procedure, SOS and COI support, and label generation during manufacturing is provided elsewhere in this document.

B. Interaction between special and built-in objects to support SOS and COI.

In fig. 3C-3E schematically illustrate the tracking of biological material during production in a manufacturing facility in accordance with some embodiments of the present invention. In some embodiments, initially, when a tumor sample obtained from a patient at a clinical site is delivered to a manufacturing facility, the shipping label is scanned and the information obtained from the scan is associated with appropriate computer objects in the system. Information may include, but is not limited to, cell order identifier, patient-specific identifier, time of tumor resection, processes used to resect the tumor, processes used to store the tumor after resection, and expected processes to be used to propagate the cell therapy product from the tumor . After the patient information obtained from the scan is verified against the cell order request that is separately accepted at the manufacturing facility (eg, through the manufacturing portal 140), the tumor is indicated in the system as accepted by the manufacturing facility and the chain of custody passes to the manufacturing facility. Details of how chain of custody and chain of identification are maintained throughout the manufacturing process are provided elsewhere in this document.

The tumor then moves on to quality control. During quality assurance, the tumor information obtained from the scan is further checked to ensure that the tumor sample meets the required quality criteria. Information verified in this process includes, for example, information related to the processes used to resect the tumor and the processes used to store and transport the tumor after resection. Information related to the processes used for tumor resection may include, for example, various fields processed from the tumor acquisition form, which is further described in detail elsewhere herein. When a tumor is deemed to meet the quality criteria, the appropriate label is scanned and the corresponding objects are updated. The updated information included in the objects is available throughout the system, including, for example, at the hospital facility, as well as at the logistics service provider (also referred to herein as the courier service). In some embodiments, various processes associated with the hospital facility and the courier service are updated based on the updated objects.

The tumor then goes into production. The Day Zero Lot Label is then scanned to ensure that the relevant tumor sample moves into production and will be processed through the appropriate manufacturing processes. The tumor is then transferred to the appropriate flask (which contains the media and reagents for the expansion process with which the patient's tumor is to be treated) and the flask label is scanned. The corresponding objects are updated with the information obtained from the scan with the corresponding timestamps.

The zero-day flask is then manually moved into the incubator room, from where it is moved to the production area. In the production facility, the flask label is scanned again and the information contained on it is recorded. Information from the flask label is also used to ensure that appropriate manufacturing processes are followed.

After the first required period of time (eg, 11 days shown in Fig. 3C), depending on the specific manufacturing process, the flask is inoculated. During the inoculation process, it may be necessary to remove the flask from the production area. In such cases, the vial label is scanned and the batch record is updated before and after the vial leaves the production facility. Pre- and post-scan information is compared before additional processes are performed.

After the inoculation process, the flask is again transferred to the production room. The flask label is scanned before and after re-transfer to the production facility after the inoculation process, and batch records are updated. Information obtained before and after scanning is compared to ensure that the correct flask is used, and appropriate additional processes are applied to further process the cell therapy product.

After the second required period of time (eg, day 16, shown in Fig. 3C), the cells from the seeded flask are divided into multiple bags (eg, 5 bags, shown in Fig. 3C). Labels for each of the bags are scanned before and after re-transfer to the production facility, and batch records and related items are updated accordingly. Information obtained before and after scanning is used to verify that the cells from the flask are separated according to the cell order request and that the bags are further processed through appropriate manufacturing processes.

- 156 045580

In some embodiments, quality analysis is performed at the first and second required times to ensure that the manufacturing process is proceeding as requested for the cell order and according to the specified schedule. In such embodiments, quality control results may also be included in the scanned information. Upon receipt of such information as a result of the relevant scans, if it is determined that the quality of the cell therapy product obtained at the relevant point in time does not meet the defined quality criteria, the batch record and related objects are updated accordingly. If necessary, the production process schedule is also updated. In some embodiments, the updated schedule may be communicated to the hospital facility as well as the courier so that the schedules of the respective processes to be performed by the hospital facility and the courier may be updated accordingly. Details about when and how the production schedule is updated are provided elsewhere in this document.

The production process then continues (either as scheduled or with appropriately modified schedules and/or intermediate steps) in multiple batches to a given end point. In some embodiments, cells from one of a plurality of packages are used to conduct quality assays on the final expanded cell therapy product. The package selected from the quality analysis, as well as the packages containing the cells to be released as the final product, are then labeled and the labels scanned to update batch records and related items.

The bags to be released as a final product are then placed into cassettes and frozen for transport to a healthcare facility. The cassette labels are scanned and the batch records are updated along with the associated items. The label for the package that will be used for quality control analyzes is then scanned, the batch record and associated objects are updated, and the package is moved to the quality control station. Once quality control results are received, the results are scanned and batch records and related objects are updated.

In some embodiments, the pouches to be released as the final product may be provided to the courier (and ultimately the hospital facility) with the caveat that the product in the pouches has not yet been approved for use in the patient's therapy because quality testing results have not yet been obtained. not available. In such embodiments, the relevant objects are updated in real time when quality analysis results are received, thereby reducing the time it takes to deliver the final cell therapy product to the patient.

Once the quality analysis results indicate that the cell therapy end product is approved for therapeutic use in a patient, the cassettes are scanned and batch records and related facilities are updated. The cassettes are then prepared for transport, handed over to the logistics provider (i.e. the courier) and scanned, indicating that the chain of responsibility has passed to the courier.

The courier then delivers the final cell therapy product to the treatment facility, where the labels are scanned again to indicate that the chain of custody has passed to the treatment facility. Batch records and related facilities are updated so that the manufacturing facility and courier are notified that the final cell therapy product has arrived at the healthcare facility.

C. Labeling of the cell therapy product during the manufacturing process and maintaining the chain of responsibility and chain of identification.

In fig. 3F schematically illustrates the process of maintaining SOC and COI throughout the journey of a cell therapy product from solid tumor production through the manufacturing process to infusion into a patient in accordance with certain embodiments of the manufacturing process (eg, GEN 2 process or GEN 3 process). In some embodiments, the hospital side interface 110 initially receives patient information from an employee (eg, a nurse) at the hospital connected to the hospital side interface 110. Patient information may include health insurance information, personal information, health-related information, patient registration information (such as consent forms), or any other patient-related information that may be useful in identifying and caring for the patient.

Once a hospital employee, such as a clinician, has identified a patient as a candidate for, for example, TIL infusion therapy, the patient may consent to continued TIL infusion therapy via a computer connected to the hospital-side interface 110.

The hospital staff member can then order TIL infusion therapy for the patient through the 120 event scheduler. The hospital billing officer may then place a purchase order for the TIL infusion therapy for the patient through the logistics interface 130. The TIL infusion therapy order and the purchase order may be transmitted to the hospital-side interface 110. The hospital side interface 110 may confirm receipt of the order and purchase order, as well as

- 157 045580 confirm patient registration and consent before issuing a request to order cells to the 120 event planner. Likewise, the hospital-side interface may communicate with the insurance provider to notify that various patient treatment events have been scheduled, and may also provide the patient's treatment event schedule to the insurance provider for payment processing.

The patient-specific identifier can then be associated with the cell order request and attached to the patient-associated biological material at each stage of processing and/or delivery to provide continuous tracking of the chain of custody and chain of identification for the biological material, as detailed below. this document. A patient-specific identifier may also be associated with the patient as well as the treatment equipment used to manage the patient's various treatment processes to track the patient throughout the treatment regimen.

The patient-specific identifier may also be communicated to the insurance provider through the hospital-side interface 110 to enable payment processing after various patient treatment events. The patient-specific identifier may optionally be communicated to the courier via the courier portal 130 so that the courier can verify the identity of the patient associated with the biological material being transported.

In various embodiments described herein, any communication between scheduling module 125 and hospital-side interface 110, manufacturing portal 140, or logistics interface 130 related to scheduling production events and/or patient treatment events (whether modified or no) is accompanied by a patient-specific identifier associated with the cell order request being processed and scheduled. Such communication, including a patient-specific identifier, allows hospital-side interface 110, manufacturing portal 140, and logistics interface 130 to accurately process, track, and identify biological material, thereby avoiding misidentification of biological material or patient and increasing patient safety.

In addition, as detailed herein, a patient-specific identifier is used to generate labels for containers used in the propagation process to enable COC/COI to be maintained during the propagation process and to allow post-hoc auditing for regulatory compliance.

In some embodiments, the patient-specific identifier may be included in the cell order identifier, which is also structured to be used for chain of identification/chain of responsibility tracking as well as event history tracking. In some embodiments, the cell order identifier, in addition to the patient-specific identifier, may include multiple characters or fields, each of which corresponds to biological material from the patient that has undergone some processing step. In fig. 3G is a representative image of a label for a cell therapy product in accordance with some embodiments. In some embodiments, the label includes a cell order identifier 350, a 2D code (eg, QR code) 362, a reason field 370, and a product field 380. The cell order identifier 350 may include various fields, such as, for example, a patient name 354, a patient-specific identifier 352, a patient's date of birth 356 (for example, for additional verification), a hospital-associated patient identifier 358, and a manufacturing facility number 360 parties.

Thus, the label allows for the maintenance of COI/COC and also allows for tracking of materials obtained from tumor resection at the clinical treatment center, delivery of tumor material to the manufacturing facility, production including testing of all in-process samples and finished products, delivery of drug product to clinical treatment center and drug infusion.

In some embodiments, different labels may be generated at different points in time along the cell therapy product's journey from solid tumor receipt through the manufacturing process to infusion into the patient, as shown in FIG. 3N. Labels generated at different points in time may include additional or different information than that included in the label shown in FIG. 3G. For example, in some embodiments, labels generated by the cell propagation process, which are, for example, affixed to containers used in the cell propagation process, may include a field corresponding to various points in time and the result of a determination made at the corresponding point in time to determine whether whether the acceptance parameters of the expanded cell therapy product meet the defined acceptance criteria.

Thus, at any stage of the process, the label provides information about the patient (via a patient-specific identifier), thereby maintaining a chain of identification and chain of responsibility. The label can also provide information about the various processing steps that the associated biological material has undergone, providing a record of the chain

- 158 045580 responsibility, as well as a history of events, for example for audit purposes.

Thus, the system 300 may be configured to maintain COI and COC throughout the cell therapy product manufacturing process and supply chain. In addition, the system 300 may be configured to include protections that comply with 21 CFR Part 11, HIPAA (Health Insurance Portability and Accountability Act) and GDPR (General Data Protection Regulation) requirements, as well as ensuring data security and protection.

To limit access to sensitive data, system 300 implements roles and user-based access in some embodiments. In some embodiments, a timestamp may be included in the label at the time it is generated.

In some embodiments, the cell order identifier tracked by the tracking module may further include characters or fields associated with, for example, various shipping/transport events, various manufacturing process events, and/or various steps in a treatment regimen. These characters or fields may not be printed on the label, but rather generated or added by the tracking module to track biological material associated with the patient and to track therapy progress. In such embodiments, a timestamp indicating the completion time of a particular step may be associated with or added to the cell order ID at each step where a given index or field is changed or updated. It should be understood that the information included in such updated cell order identifier may also be used to dynamically reschedule a patient's treatment events, as described in detail elsewhere in this document.

1. Labeling of the cell therapy product during the production process.

As shown in FIG. 3F, in some embodiments, the cell order ID is printed on readable labels and linked to barcodes or QR codes (or 2D codes) to provide adequate verification of identification from tumor resection to autologous drug infusion. The label can be applied to objects associated with biological material, as well as with a treatment regimen. For example, a label containing a patient-specific identifier may be affixed to a container in which a resected tumor is transported from a hospital or clinical site to a TIL manufacturing facility, TIL manufacturing equipment and containers for TILs at various stages of production, a shipping container for transporting expanded TIL back to the hospital or clinical facility, equipment associated with various treatment events, or any combination thereof.

For example, in some implementations, a patient may log in through the patient access module 116 of the hospital-side interface 110 and register. Once a patient's trackable information is registered and collected into the system, the system can assign a patient-specific identifier (COI number) to the patient. Assigning a patient-specific identifier allows you to schedule a resection date and production interval.

Planning a production interval in the system triggers the generation of a cell order and assignment of a lot number at the production facility.

Prior to tumor resection, the clinical site can access the system, confirm correct patient identification, and generate a tumor vial label and tumor shipper label. The label may include a patient-specific identifier in addition to the cell order identifier. In some embodiments, the patient-specific identifier may be a field in the cell order identifier.

After tumor resection, a chain of responsibility is initiated in tracking module 112. The labeled tumor vial can then be placed in a vehicle and handed over to a courier for transport to the production site. The chain of responsibility continues as the courier checks that the cell order number on the shipper's label matches the cell order number on the delivery order in the courier's system, which is integrated into the system 300, thereby providing real-time tumor tracking between the treatment center and the manufacturing facility. object.

Once the tumor is delivered to the manufacturing facility, a delivery confirmation can be generated in the system. Once the tumor is received, production personnel enter the production lot number into the system in the production module 140 through the COC/COI module 144. Upon retrieval of the patient's medical record (accessible through production coordination module 118), the tumor vial label is scanned into the system to ensure that the patient-specific identifier is associated with the production lot number. After verification, the SOS is transferred to the production facility.

Manufacturing plant personnel can log into the system and the labeling module 142 allows the generation of work-in-process (WIP) labels that contain a cell order request. To facilitate auditing of records after the fact, WIP labels can be affixed to the lot and cell culture flask records. In fig. 3G is a representative image of a WIP label in accordance with an embodiment. In fig. 3H shows various types of WIP labels and information included in each type of label, in accordance with an embodiment.

- 159 045580

Production personnel can then scan labels at key transition points during the manufacturing process to ensure that the cell order ID number on the cell culture flask matches the batch record, ensuring COI is maintained throughout the manufacturing process.

In addition, each quality control (QC) sample will have its own COI number label to ensure that the results obtained are then linked to the appropriate batch record. Upon completion of the manufacturing process, labeling module 142 may generate finished product labels. In fig. 3I and 3J show examples of finished product labels.

As shown in FIG. 3I-3J, these finished product labels contain unique patient identification, including name 354, date of birth (DOB) 356, and patient-specific identifier 352. Finished product labels are affixed to the final product, and the batch record and labels are scanned to ensure COI and a complete chain of custody within the manufacturing plant.

The finished product is transferred to the shipper with the same cell order ID, including the corresponding patient-specific ID, verified, and transferred to the courier for delivery to the treatment center for infusion. The courier can then verify that the cell order ID and patient-specific ID on the shipping container match the cell order and patient information in the courier system received through the logistics interface 130, thereby providing near real-time tracking of each drug lot. , between the production facility and the treatment center. In this case, the SOS is transferred from the production facility to the courier.

The courier delivers the finished product to the treatment center for infusion into the patient's body. The courier may initiate a proof of delivery (POD) through the logistics interface 130, and the SOC is transmitted from the courier to the treatment center. The COI ends with the infusion of the product into the patient's body at a treatment center.

As described herein, passing a patient-specific identifier between hospital-side interface 110, event scheduler 120, manufacturing portal 140, and logistics interface 130 after each processing step ensures that all parties involved (e.g., clinical site, manufacturing facility, and logistics provider services) obtain a record of the chain of responsibility as well as the chain of identification of the biological material, and also obtain a record of the various process steps through which the biological material has passed.

The patient-specific identifier and cell order request provided for solid tumor via a label thus allows delivery tracking to maintain the chain of custody and chain of identification that are necessary for patient safety as well as regulatory compliance (e.g., FDA audits ).

2. Reconciliation of labels in case of changes in the production process.

Once a solid tumor has been accepted, the manufacturing facility begins production of the cell therapy product according to the cell order request. For example, at least a portion of a solid tumor may be used to produce a cell therapy product using cell expansion techniques.

In some embodiments, a computer subsystem associated with a manufacturing facility, such as labeling module 142, may cause a second label to be printed associated with a portion of the solid tumor used for cell propagation. The second label may include a patient-specific identifier and a cell request identifier. The second label also allows for traceability of the cell therapy product to maintain chain of custody and chain of identification.

Acceptability parameters of the manufactured cell therapy product are then determined at various points in time during manufacturing of the cell therapy product. The acceptability parameters are then compared in the acceptance determination module 123 to determine whether the acceptability parameters determined at a particular point in time correspond to the acceptance criteria at the corresponding point in time. Acceptability parameters may be determined by any suitable method or process described herein. Additionally, the acceptance criteria may be any of the acceptance criteria described herein.

In some embodiments, the result of determining the acceptance parameters and whether the acceptance parameters meet the acceptance criteria may be added to the cell order identifier as described in other sections herein. In some embodiments, a third label may be generated based on the result of determining whether the acceptance parameters meet (or fail to meet) the acceptance criteria.

For example, if the acceptance parameters at the second time point do not meet the acceptance criteria at the second time point, a third label is generated for the container used in the manufacturing process, including a reason code to communicate that the acceptance parameters at the second time point do not meet the criteria acceptability at the second point in time and why the eligibility criteria at the second point in time are not met.

In addition, if it is determined that the cell therapy product obtained at the second time point can be reprocessed from the first time point prior to the second time point to properly obtain a cell therapy product that meets the acceptance criteria at the second time point, the third label may include information such as, for example, a new or updated Cell Request ID as described elsewhere in this document. In such cases, the third label may further include a reason code to convey information regarding the acceptance criteria at the second time point and why it was appropriate to reprocess the cell therapy product from the first time point.

Next, depending on whether the acceptance criteria are met at one or more points in time, as determined in the acceptance determination module 123, the production schedule for the production of the cell therapy product is modified accordingly to generate an updated production schedule, as described in other sections herein. In addition, the availability of production slots at the manufacturing facility is also modified accordingly to reflect changes, if any, in the current production schedule of the cell therapy product. The changed or updated availability of production slots at that production plant can then be transmitted to the computing device. In addition, the patient's preliminary treatment event schedule may also be modified based on the updated production schedule, and the patient's updated treatment event schedule may be transmitted to the computing device.

Manufacturing of the cell therapy product is then completed according to the updated production schedule.

In some embodiments, a production label is generated corresponding to each of a plurality of time points at which acceptance parameters are determined. The manufacturing label may, at any given time, include updated information related to the quality of the cell therapy product being manufactured. The updated information may include eligibility parameters at the relevant point in time and/or whether the eligibility parameters meet the acceptance criteria at that point in time.

Additionally, in some embodiments, the controller controlling the manufacturing process may be configured to read the updated manufacturing label and determine a subsequent processing step. For example, if an updated manufacturing label after quality control at a second time point indicates that the cell therapy product does not meet certain acceptance criteria, but the cell therapy product can be reprocessed from the first time point, the controller can make appropriate changes to the manufacturing process. In addition, based on the information on the updated label, appropriate changes are also made to the production schedule, availability of production slots, and the patient's treatment event schedule. Those skilled in the art will appreciate that the updated manufacturing label may include a cell order identifier and a patient-specific identifier in addition to information related to the quality of the cell therapy product being manufactured and a reason code to facilitate maintaining a chain of identification and chain of responsibility.

Those skilled in the art will also appreciate that information related to changes in production schedules, production slot availability, and patient treatment event schedules resulting from reading updated production labels can be communicated to the hospital-side interface and logistics interface to allow the corresponding computing devices to make appropriate changes to the corresponding schedules associated with these objects.

D. Coordination of cell therapy product production with patient treatment events.

In fig. 4A and 4B show a flow diagram of a method for coordinating the production of TILs for a patient in accordance with an embodiment of the present invention. In some embodiments, the method shown in FIG. 4A and 4B is implemented in the overall system depicted in FIG. 3A, and in some embodiments, the method shown in FIG. 4A and 4B is implemented by parts of the system shown in FIG. 3A. In some embodiments, the event scheduler 120 receives, for example, at step S305, a request to order cells for expansion of a cell therapy product, such as T cells, for a patient from the hospital-side interface 110, and a patient-specific identifier associated with the order request cells.

In some embodiments, the patient-specific identifier or, alternatively, the cell order identifier is generated by the event scheduler 120, for example, at step S310, rather than by the tracking module 112.

The cell order request may include information such as, for example, patient identifying information, target dates for various patient treatment events, and/or target parameters for the final expanded TIL product. After receiving a request to order cells plani

- 161 045580 Event manager 120 may send an acknowledgment to hospital-side interface 110 indicating that the cell order request corresponding to the patient-specific identifier has been accepted. In embodiments in which the patient-specific identifier is generated by the event scheduler 120, the event scheduler 120 may transmit an acknowledgment indicating that the request to order cells for the patient has been accepted and provide the patient-specific identifier or cell order identifier to the interface 110 at side of the hospital. In addition, the event scheduler 120 may transmit a target schedule of patient treatment events based on the received cell order request.

In addition, the event scheduler 120 may transmit an initiation request including a patient-specific identifier, for example in step S315, to a hospital-side interface 110, such as a clinical facility, to perform a procedure on a patient to obtain a solid tumor from the patient and schedule a delivery time. courier of the received solid tumor to the production facility. As described herein, the procedure may include, but is not limited to, a tumor biopsy to remove a portion of the tumor from the patient's body. In some embodiments, the event scheduler 120 may receive a procedure date from an employee using the hospital-side interface 110. In response, the event scheduler 120 may prompt the employee to order supplies (eg, a tumor resection kit, a tumor shipping container, or a cryotransport container) as needed and associate them with a patient-specific identifier.

After performing the procedure for obtaining a solid tumor and sending the solid tumor, for example, in step S320, and after receiving the obtained solid tumor at the production plant, the scheduling module 125 dynamically schedules, for example, in step S325, various production events as well as various patient treatment events and associates each event with a patient-specific identifier. In some embodiments, scheduling module 125 determines a schedule of patient production events and treatment events prior to receiving a solid tumor acquisition, such as after receiving solid tumor acquisition scheduling information from a patient from the hospital-side interface 110, and associates each event with a patient-specific identifier .

The schedule of various production events is associated with a patient-specific identifier and may include corresponding target dates on which various production steps are performed (eg, steps A-F shown in Figures 2A, 2B or 2C and/or 9), as well as a corresponding target completion date TIL production process. As described herein, target times for various production steps may depend on factors such as the size of the resulting tumor, the number of cells before the start of a given step, the numerical fold at the end of a given step, the number of days required to achieve a certain numerical fold at a given stage, and /or other factors.

The patient's various treatment event schedule may include target dates for various patient treatment events associated with a patient-specific identifier, such as, for example, a TIL infusion target date, a lymphodepletion date, a length of hospital stay, a schedule for an IL-2 infusion regimen, and/or other similar dates related to treatment. As described herein, the patient's treatment event schedule is dependent on the production event schedule and, in particular, the TIL production completion date. In some embodiments, a schedule of various patient treatment events may be provided to the hospital-side interface 110 along with a patient-specific identifier.

The scheduling module 125 is configured to dynamically change the schedule of production events as well as the schedule of patient treatment events based on the progress and success of various production steps. For example, depending on whether the acceptance parameters associated with the expanded cell therapy product at different stages meet the defined acceptance criteria, the start dates for subsequent manufacturing steps need to be changed, which in turn changes the target completion date, and consequently the dates The patient's treatment events must be modified using, for example, the methods depicted in FIG. 4B or 4C.

Acceptance determination module 123 determines whether the acceptance parameters associated with the expanded cell therapy product meet the determined acceptance criteria. Acceptability parameters include, but are not limited to, cell count, cell viability, sterility, mycoplasma count, CD3 count, CD5 count, interferon-γ (INF-γ) production, endotoxin assay result, Gram stain assay result, etc. Some or all of these acceptability parameters may need to be met according to the acceptance criteria depending on the stage at which the acceptability parameters are measured. For example, at the end of the first initiation propagation (also referred to herein as the first time point), for example, 11 days after the start of propagation, the acceptance criterion may be that the number of viable cells must be at least 5x10 ⁶ . The acceptance criteria at different stages also depend on the process (eg Gen 2, Gen 3, Gen 3.1, etc.) used to perform the propagation.

Thus, the acceptance determination module 123 can determine whether the acceptance parameters meet certain acceptance criteria at more than one different point in time after the propagation is initiated depending on the process used to perform the propagation. In other words, the acceptance determination module 123 performs a quality test, also referred to herein as a quality check, at each of the various predetermined time points to determine a subsequent course of action, and the planning module 125 dynamically schedules subsequent production steps and patient treatment events based on the results of the quality check. received from the acceptance determination module 123.

One example of acceptance parameters and acceptance criteria for end product testing for a Gen 2 process similar to Gen 2 and Gen 3 is given in Table. IN.

Table B

Test type (acceptability parameter) Way Eligibility Criteria Control of finished products Cell viability Fluorescence at least 70% Total viable cells Fluorescence from 1е9 to 150е9 Identity (%CD45+/CD3+) Flow cytometry Similar Gen 2: at least 90% CD45+CD3+ TIL for non-ovarians Gen 3: at least 90% CD45+CD3+ TIL for non ovaries at least 85% CD45+CD3+ TIL for ovaries Production of gamma interferon (stimulated unstimulated) Stimulation and ELISA more than 500 pg/ml

Referring to FIG. 4B, in one embodiment, after initiation of expansion of the cell therapy product, the acceptance determination module 123 determines, for example, in step S402, whether the expanded cell therapy product at the first time point has passed the quality check associated with the first time point (for example, after step B shown in Fig. 2A and/or 9). After determining that the items have failed the quality check at the first time, for example, in step S404, the acceptance determination unit 123 determines whether the items have failed the quality check due to contamination. If the cells fail quality control due to contamination, the case is considered for possible termination, for example at step S422.

On the other hand, if the cells fail quality control for reasons other than contamination, such as low cell count or low viability, scheduling module 125 may extend the current step by a time depending on the reasons why the cells failed quality control, and review the results of the final cell collection, for example, at step S408. In some embodiments, all subsequent production steps and patient treatment events may be rescheduled by extending the current step for some time.

Alternatively, scheduling module 125 may reschedule only patient treatment events to account for a possible delay in completion of the manufacturing process or to account for possible cryogenic freezing of the manufactured cell therapy product to allow for a time delay between completion of manufacturing and infusion of the cell therapy product. It should be understood that in some embodiments, the cell therapy product contains T cells from a patient. In some embodiments, the cell therapy product comprises tumor infiltrating lymphocytes (TILs). Accordingly, the description herein may

- 163 045580 ease of referring to the cell therapy product interchangeably as T cells or TILs.

For example, in some cases, a cell therapy product may pass final quality control even though it failed quality control at the first time point. In such cases, it may be prudent to deviate from the optimal schedule of production and patient treatment events and defer lymphodepletion until final quality control is performed to avoid unnecessary patient treatment. Thus, depending on the reasons why the cells failed the quality check at the first time point, it is determined in step S418 whether the patient's treatment event schedule should deviate from the patient's optimal treatment event schedule. Upon determining that the patient's treatment event schedule should deviate from the optimal schedule, scheduling module 125 may reschedule (i.e., postpone), for example, at step S420, lymphodepletion, as well as subsequent patient treatment events, and additionally optionally schedule a cryogenic phase. freezing after completion of the production process.

Returning to step S402, if the cells pass quality control at the first time point, no changes to the manufacturing process schedule or patient treatment events may be required. Then, the acceptance determination module 123 determines, for example, in step S406, whether the expanded T cells at the second time point pass the quality check associated with the second time point (for example, after step C or step D shown in Fig. 2A and/or 9 ).

If the cells fail the quality test at the second time point, the acceptance determination unit 123 determines, for example, in step S404 whether the cells fail the quality test due to contamination. If the cells fail quality control due to contamination, the case is considered for possible termination, for example at step S422.

On the other hand, if the cells fail quality control for reasons other than contamination, such as low cell count or low viability, scheduling module 125 may extend the current step by a time depending on the reasons why the cells failed quality control, and review the results of the final cell collection, for example, at step S408. In some embodiments, all subsequent production steps and patient treatment events may be rescheduled by extending the current step for some time.

Alternatively, scheduling module 125 may reschedule only patient treatment events to account for a possible delay in completion of the manufacturing process or to account for possible cryogenic freezing of the produced TILs to allow for a time delay between completion of manufacturing and infusion of the TILs.

For example, in some cases, cells may pass the final quality check even though they failed the quality check at the second time point. In such cases, it may be prudent to deviate from the optimal target schedule (also referred to herein as the golden mean) of the patient's treatment events and defer lymphodepletion until final quality assurance is performed to avoid unnecessary treatment of the patient. Thus, depending on the reasons why the cells did not pass the quality check at the second time point, the scheduling module 125 may reschedule (i.e., postpone), for example, at step S420, lymphodepletion, as well as subsequent patient treatment events, and additionally plan for a cryogenic freezing step after completion of the production process.

In some embodiments, when cells fail quality control at the first or second time point, the patient-specific identifier may be updated to identify that the cells failed quality control at the appropriate time point, and hospital-side interface 110 and logistics interface 130 are notified that there is a possibility that patient treatment events (associated with a patient-specific identifier) may have to be canceled or postponed. If the patient's treatment event schedule has deviated from a predetermined schedule (e.g., a happy medium schedule) and the patient's treatment event schedule has been changed, the hospital-side interface 110 and the logistics interface 130 are notified that the schedule corresponding to the patient-specific identifier has been updated, and the updated the schedule is transmitted to the hospital-side interface 110 and the logistics interface 130.

Such notification allows the clinical site or hospital to appropriately adjust various schedules of patient treatment events, as well as make necessary logistical arrangements regarding the availability of appropriate personnel and the availability of facilities and equipment necessary for the patient's respective treatment events. The notification may further make it easier for the hospital or clinical site to inform the patient of changes in the patient's treatment event schedule. Alternatively, the event scheduler 120 and/or the hospital-side interface 110 may communicate with the patient directly to notify the patient of a change in the patient's treatment event schedule and allow the patient to coordinate with the hospital or clinical site to arrange the patient's treatment events according to the changed schedule. .

In some embodiments, the hospital-side interface 110 may optionally

- 164 045580 communicate the updated schedule to the insurance provider so that appropriate arrangements can be made to process the payment.

Likewise, the notice allows the logistics provider(s) to take the necessary steps to reschedule the delivery of the biological material, such as, for example, stowing appropriate shipping crates and ensuring that appropriate personnel are available to handle the biological material.

Returning to step S406, if the cells pass the quality check at the second time point, the scheduling module 125 may maintain a schedule of patient treatment events. For example, in some embodiments, scheduling module 125 may communicate, for example at step S410, to hospital-side interface 110 that a patient may be scheduled for lymphodepletion treatment based on a predetermined schedule with the caveat that there is a low but non-zero probability that the manufacturing process may still be delayed or may not produce the desired end product. Such a connection is indicated in Fig. 4B how lymphodepletion is at risk. No changes are made to the patient's production schedule or treatment events in this situation.

Then, the acceptance determination unit 123 determines, for example, in step S412, whether the expanded T cells at the third time point pass the quality check associated with the third time point (for example, after step D or step E shown in Fig. 2A and/or 9 ).

If the cells pass quality control at the third time point, the expanded cells are considered ready for product release on the target TIL infusion date. In such cases, the scheduling module 125 notifies, for example, in step S424, the hospital-side interface 110 that the patient's treatment events should continue according to the target schedule. In other words, no changes are made to the schedule.

On the other hand, if the cells do not pass the quality check at the third time point, a product impact assessment (eg, by a physician or primary care provider) is performed, for example, at step S414, to determine whether the expanded cells can indeed be provided for infusion or treatment should be stopped depending on the reasons why the cells failed quality control at the third time point. If, after assessing the impact of the product, it is determined, for example at step S416, that treatment can be continued with the caveat that there may be some risk associated with continuing treatment with the available product, the expanded cells are approved for release, for example at step S424, without any changes in the patient's treatment event schedule.

Referring to FIG. 4C, in one embodiment, after initiation of expansion of the cell therapy product, the acceptance determination module 123 determines, for example, in step S402, whether the expanded cell therapy product at the first time point has passed the quality check associated with the first time point (for example, after step B shown in Fig. 2A and/or 9). After determining that the items have failed the quality check at the first time, for example, in step S404, the acceptance determination unit 123 determines whether the items have failed the quality check due to contamination. If the cells fail quality control due to contamination, the case is considered for possible termination, for example at step S422.

On the other hand, if the cells fail quality control for reasons other than contamination, such as low cell count or low viability, scheduling module 125 may extend the current step by a time depending on the reasons why the cells failed quality control, and review the results of the final cell collection, for example, at step S408. In some embodiments, all subsequent production steps and patient treatment events may be rescheduled by extending the current step for some time.

Returning to step S402, if the cells pass quality control at the first time point, no changes are made to the manufacturing process schedule or patient treatment events. Then, the acceptance determination module 123 determines, for example, in step S406, whether the expanded T cells at the second time point pass the quality check associated with the second time point (for example, after step C or step D shown in Fig. 2A and/or 9 ).

If the cells fail the quality check at the second time point, the acceptance determination unit 123 determines, for example, in step S404' whether the cells fail the quality check due to contamination. If the cells fail quality control due to contamination, the case is considered for possible termination, for example at step S422.

On the other hand, if the cells fail the quality test for reasons other than contamination, such as low cell count or low viability, the acceptance determination unit 123 determines, for example, in step S458, whether the cell therapy product obtained in the second a point in time of sufficient quality to enable the cell production process to be repeated from the first point in time to produce a cell therapy product at a second point in time with acceptance parameters that meet the acceptance criteria at the second point in time. Such a determination may be made based on the acceptability parameters of the cell therapy product obtained at the second time point. For example, if the parameters

- 165 045580 acceptance at the second time point meets all the acceptance criteria at the second time point except for the total number of viable cells, it may be advisable to repeat the cell production process between the first and second time points to obtain the required number of viable cells.

If it is determined that it is not practical to perform the cell production process again from the first time point, the case is considered for possible termination, for example, in step S422.

On the other hand, if it is determined that re-executing the cell production process between the first and second times is appropriate, the scheduling unit 125 estimates the completion time of the cell production process after re-executing the process between the first and second times, for example, in step S460. It should be understood that the time required to re-perform the cell production process between the first and second time points can be estimated based on the acceptance parameters of the cell therapy product obtained at the second time point. Thus, the time required to complete the cell expansion process, including repeating the process from the first time point, to produce the cell therapy product may depend on the acceptance parameters of the cell therapy product obtained at the second time point.

Scheduling module 125 may then reschedule all subsequent manufacturing steps and patient treatment events by re-executing the cell manufacturing process from the first time point based on the acceptance parameters of the cell therapy product obtained at the second time point. For example, scheduling module 125 may reschedule patient treatment events to account for a delay in completion of the manufacturing process or to account for possible cryogenic freezing of produced TILs to allow for a time delay between completion of manufacturing and infusion of the TILs.

On the other hand, if the acceptance parameters of the cell therapy product obtained at the second time point meet all acceptance criteria, the cell production process can continue according to the original schedule.

Those skilled in the art will appreciate that even after re-performing the cell production process from the first time point, when determining that such re-performing is appropriate, determine the acceptability parameters of the cell therapy product produced at the repeated second time point (also referred to herein as alternative second point in time). It is then again determined whether the acceptance parameters at a repeated second time point (which may be different from the initial second time point in the cell production process) meet the second time point acceptance criteria before continuing the cell production process after the second time point.

The ongoing process then follows the same path B as shown in FIG. 4B in some embodiments. For example, in some cases, cells may pass the final quality check even though they failed the quality check at the second time point. In such cases, it may be prudent to deviate from the optimal target schedule (also referred to herein as the golden mean) of the patient's treatment events and defer lymphodepletion until final quality assurance is performed to avoid unnecessary treatment of the patient. Thus, depending on the reasons why the cells did not pass the quality check at the second time point, the scheduling module 125 may reschedule (i.e., postpone), for example, at step S420, lymphodepletion, as well as subsequent patient treatment events, and additionally plan for a cryogenic freezing step after completion of the production process.

Those skilled in the art will appreciate that when a cell production process has multiple points in time to determine acceptance parameters and whether those acceptance parameters meet the acceptance criteria at those respective times, it may be advisable to re-perform steps of the cell production process from the immediately preceding one. moment in time. Thus, the terms first time point and second time point do not necessarily describe numerically the first and numerically second time points at which the acceptability parameters are determined. Instead, the first time point refers to a point in time in the cell production process from which it may be appropriate to repeat steps of the cell production process if the acceptance parameters at a subsequent time point do not meet the acceptance criteria at that time point. For example, in Process 1C (see FIG. 6), the first and second time points may be day 27 and day 30, respectively, day 30 and day 36, respectively, or day 36 and day 43, respectively. Similarly, in process 2A (see FIG. 6), the first and second time points may be day 11 and day 16, respectively, or day 16 and day 22, respectively.

In some embodiments, when cells fail quality control at the first or second time point, the patient-specific identifier may be updated to identify that the cells failed quality control at the appropriate time point and the hospital-side interface 110 and the logistics interface 130 are notified that there is a possibility that the patient's treatment events (associated with a patient-specific identifier) may have to be canceled or postponed.

Because the production schedule for a cell therapy product may deviate from the optimal schedule if re-executing the cell production process at the first time point is appropriate, the patient's treatment event schedule also deviates from the desired schedule (eg, a happy mean schedule). Accordingly, the scheduling module reschedules the patient's treatment events, and the hospital-side interface 110 and logistics interface 130 are notified to update the schedule corresponding to the patient-specific identifier. The updated schedule is transmitted to the hospital-side interface 110 and the logistics interface 130.

As described herein, such scheduling change notification allows the clinical site or hospital to appropriately adjust the patient's various treatment event schedules. In addition, necessary logistical arrangements may be made regarding the availability of appropriate personnel and the availability of facilities and equipment necessary for the patient's respective treatment events. The notification may further make it easier for the hospital or clinical site to inform the patient of changes in the patient's treatment event schedule. Alternatively, the event scheduler 120 and/or the hospital-side interface 110 may communicate with the patient directly to notify the patient of a change in the patient's treatment event schedule and allow the patient to coordinate with the hospital or clinical site to arrange the patient's treatment events according to the changed schedule. .

In some embodiments, the hospital-side interface 110 may further communicate the updated schedule to the insurance provider so that appropriate action can be taken to process the payment.

Likewise, the notice allows the logistics provider(s) to take the necessary steps to reschedule the delivery of the biological material, such as, for example, stowing appropriate shipping crates and ensuring that appropriate personnel are available to handle the biological material.

In some embodiments, if treatment is not terminated, scheduling module 125 communicates with logistics interface 130 to provide a pick-up order based on a completion date determined based on quality check results at various points in time. Logistics interface 130 then communicates with a logistics service provider (not shown) to make appropriate arrangements for timely collection and delivery of the expanded TILs to a hospital or clinical site for subsequent patient treatment (eg, infusion). For example, in some embodiments, a logistics provider may need to ship a specialized container for transport of a biological sample to a hospital or clinical facility a certain number of days before the patient's scheduled treatment event.

Likewise, if it is determined that the expanded cells pass the quality check, the scheduling module 125 transmits the scheduled completion date and the patient's treatment event schedule (ie, the updated schedule) to the hospital-side interface 110. On the other hand, if it is determined that the treatment should be stopped, the scheduling module 125 informs the hospital-side interface 110 that the treatment should be stopped.

In some embodiments, when a patient's treatment event schedule changes, the patient's updated treatment event schedule is sent to the hospital-side interface 110 along with an associated patient-specific identifier.

In some embodiments, the association between the cell order identifier and the patient-specific identifier may be updated if it is determined that repeating the steps of the cell production process from the first point in time is appropriate. For example, upon determining that reexpanding the cell therapy product from the first point in time is appropriate, the cell order identifier is separated from the patient-specific identifier. A new cell order identifier associated with the cell order request may be generated, with the patient-specific identifier associated with the new cell order identifier. In some embodiments, a new cell order ID may be generated based on the eligibility parameters determined at the second time point. The cell order identifier, in some embodiments, may include fields corresponding to each time point at which the cell therapy product's eligibility parameters are determined. Additionally, in some embodiments, the cell order identifier may also include the result of determining whether the eligibility parameters meet the acceptance criteria, including, for example, which eligibility parameters meet the acceptance criteria and the value of the acceptance parameters.

In some embodiments, the patient-specific identifier, the new cell order identifier, and the estimated completion time of propagation of the cell therapy product are transmitted to the hospital-side interface so that the hospital-side interface can track the cell therapy product and associate the cell therapy product with the patient.

Various fields included in the cell order identifier may allow scheduling module 125 to determine a new schedule of patient treatment events, as well as shipping and logistics events associated with the patient treatment events, based on the updated or new cell order identifier generated at various points in time. The new schedule is then sent to the logistics interface along with the corresponding patient-specific identifier. In some embodiments, the patient-specific identifier and the updated or new cell order identifier may also be transmitted to the logistics interface along with the new schedule to maintain a chain of custody and chain of identification for the cell therapy product.

In some embodiments, if it is determined that it is not practical to perform the steps of the cell production process again, for example, because the acceptance parameters at the second time point do not meet a certain threshold for the acceptance criteria, subsequent treatment of the patient up to the second time point may be cancelled. In such embodiments, the patient's treatment cancellation information is communicated to the hospital-side interface and the logistics interface. In some embodiments, the expanded cell therapy product may be destroyed when it is determined that repeating the steps of the cell production process is not practical. For example, in cases where obtaining a viable expanded cell therapy product may not be possible, if steps in the cell production process cannot be repeated, infusion of the cell therapy product becomes impossible. Thus, it may be detrimental to the patient (e.g., either in terms of health outcomes or financial outcomes, or both) for the patient's treatment events to continue beyond the second point in time. In addition, if, based on certain eligibility parameters at the second time point, it is determined that the expanded cell therapy product at the second time point cannot be used further (for example, if the cell therapy product at the second time point is contaminated or does not meet some other acceptance criteria), the product cell therapy may be destroyed. In such cases, the cell order ID is separated from the patient-specific ID.

E. Coordination of production intervals between production plants.

In another aspect of the present invention, a method for producing a cell therapy product for a patient is described. In some embodiments, the method includes receiving a request to order cells to produce a cell therapy product for a patient. A request to order cells may be received at a computing device associated with a clinical site. A request to order cells may be received, for example, through the hospital-side interface 110. In some embodiments, the computing device may generate a patient-specific identifier and cell order identifier upon receiving a cell order request, and associate the patient-specific identifier and cell order identifier with the cell order request.

In addition to a request to order cells, the computing device may receive information regarding production intervals at a plurality of manufacturing facilities for the production of a cell therapy product. For example, a computing device may be communicatively coupled to computer subsystems associated with a plurality of manufacturing facilities via, for example, the Internet, such that the computing device may receive information related to production intervals in response to a request from the computing device. Production intervals for a relevant manufacturing facility may indicate the availability of equipment and personnel at that manufacturing facility to enable that manufacturing facility to manufacture the cell therapy product in accordance with the cell order request.

The computing device may further receive a preliminary schedule of patient treatment events for treating the patient with a cell therapy product. The preliminary schedule may be determined based on the optimal production schedule for the cell therapy product, assuming that the cell therapy product meets quality assurance criteria at each manufacturing step during the manufacturing process, as described elsewhere in this document.

Upon receiving a request to order cells, production slots, and the patient's preliminary treatment event schedule, the computing device may determine and display in a scheduling user interface a plurality of available production slots for manufacturing the cell therapy product based on the patient's preliminary treatment event schedule. Available manufacturing windows are determined based on factors such as the location of the clinical site, the location of the relevant manufacturing facilities, the availability of logistics providers to ship the cell therapy product between the relevant manufacturing facility and the clinical site, the availability of medical personnel at the clinical site for performing necessary treatment procedures related to the patient's treatment events, and any other factors that may affect the time of arrival and/or application of the cell therapy product at the appropriate location.

One of the available production slots is then selected either by the operator of the computing device or automatically by the computing device. By selecting an available manufacturing interval, the solid tumor can be obtained from the patient, for example, in a medical facility, by performing an appropriate procedure. The procedure can be performed according to a preliminary schedule and based on the available production window, taking into account delivery times and the availability of a logistics provider to deliver the solid tumor to the appropriate manufacturing facility. In some embodiments, the computing device may initiate printing of a first shipping label (see, for example, line 1 of FIG. 3H) associated with the solid tumor. The first shipping label may include a patient-specific identifier and a cell order identifier. In some embodiments, the first label may further include information related to the clinical site, the selected manufacturing facility, and the logistics provider. In addition, the first product label (see, for example, line 2 in Fig. 3H) must be associated with the product container. An example of a first product label is shown in FIG. 3G.

The solid tumor is then transported to the selected manufacturing facility according to the available manufacturing window. Those skilled in the art will appreciate that since a solid tumor comprises living cells, the timing of arrival of the solid tumor at the selected manufacturing facility must be carefully coordinated with the availability of the manufacturing window so that the manufacturing steps can be initiated within an acceptable time window. Therefore, the delivery of solid tumors from the clinical site to the selected site must be carefully coordinated based on the availability of the logistics provider as well as other factors that may affect such delivery. For example, there may be weather or traffic delays that can be expected in some cases and therefore the delivery schedule may be adjusted accordingly. In other cases, delivery delays may be unpredictable. However, even in such cases, delivery of the solid tumor from the clinical site to the selected manufacturing facility can be coordinated by other suitable means.

F. Tumor acquisition protocol.

As shown in FIG. 3A, the hospital-side system interface 300 includes a tumor acquisition module 114 that allows clinical facility personnel to obtain a solid tumor from a patient according to a predetermined protocol and enter information related to the procedure for obtaining a solid tumor from a patient.

In some embodiments, acquisition module 114 includes tumor acquisition forms that are used by personnel at a clinical site to ensure that proper procedure is followed in the process of obtaining a solid tumor (or fragment thereof) from a patient and preparing it for transport to a manufacturing facility. In fig. ZK-ZR provides illustrative screenshots of tumor acquisition molds in accordance with some embodiments of the present invention. Tumor collection forms may, in some embodiments, require the practitioner receiving the tumor from the patient to enter information relevant to the procedure being performed. The information entered can be used to update batch records for audit after the fact, if necessary.

Additionally, because information included in batch records is available throughout the system (with appropriate permissions), information related to the tumor acquisition process is also available to personnel at the manufacturing facility. In some embodiments, personnel at the manufacturing facility may have access to information related to the tumor acquisition process as a quality control measure to ensure that the biological material obtained at the manufacturing facility is suitable for further processing.

In some embodiments, tumor retrieval molds include smart mold features that require certain criteria to be met before the practitioner can proceed to the next step of the process. For example, the specialist may be required to enter information related to cell culture media, such as the expiration date applied during the process, and if the expiration date is prior to the current date, the tumor acquisition module 114 may alert the specialist. In addition, the tumor acquisition module 114 may prohibit the technician from entering any additional information related to the process, thereby forcing the technician to abort the process.

Similarly, tumor acquisition forms may require the practitioner to complete certain steps or procedures before allowing entry of information related to the subsequent step or procedure. Thus, acquisition module 114 requires the practitioner to follow a specific protocol without omitting or skipping certain steps.

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In some embodiments, the receiving module 114 may require that tumor receiving forms be verified before allowing the resulting tumor to be released to a courier for transport to the manufacturing facility. The verification requirement acts as a fail-safe system to ensure that appropriate protocols and procedures are followed when obtaining a tumor. In some embodiments, the acquisition module 114 may require that the person validating the tumor acquisition forms be different from the person entering information into the tumor acquisition forms. This verification requirement also ensures compliance with relevant regulatory requirements.

G. Patient Support Services.

Embodiments of the present invention further provide the ability to provide access to patient support services that interact with a patient or patient representative (hereinafter collectively referred to as the patient) regarding travel, health management, health insurance, reimbursement, and other treatment-related services. Patient support services may, in some embodiments, interface with a telephone interface and profile from which the manufacturing and COC/COI process (with appropriate permissions) can be accessed. In the context of this document, the term profile refers to the type of user. This profile is granted a certain level of access to the system and information in the system, as well as access to certain functions. Profiles in the system include, but are not limited to, Community Representative, Tissue Acquisition Specialist, Manufacturer, Patient Support, Patient, and Health System User.

The Community Representative profile refers to community users and is available to the center staff providing treatment to the patient. Users with a community member profile may include, for example, cell therapy coordinators, bone marrow transplant nurses, hospital billing staff, etc. Features associated with users with the Community Member profile include, but are not limited to, registering a new patient, updating patient registration information, creating and submitting a TIL order request for a patient, scheduling or changing a resection date, viewing sweet spot dates, viewing cutoff delivery dates Product, Upload Hospital Receipt Orders, Upload Patient Consent Forms, Register Patient for Patient Support Options, View and Update Caregiver Records, Complete and Submit Tumor Receipt Forms for Approval, Approval of Tumor Receipt Forms, Print Forms for tumor retrieval, loading tumor retrieval forms, and printing media bottle labels and tumor shipping labels.

Users associated with the manufacturer profile include tumor acceptance personnel, quality control personnel, manufacturing process personnel, and final product packaging personnel. Features associated with the manufacturer profile include, but are not limited to, entering lot numbers, viewing available and reserved production slots, in-process printing and scanning, end product labels, and shipping labels.

Users associated with the tumor specialist profile include, for example, surgeons and bone marrow transplant nurses. Functions associated with the Tumor Acquisition Specialist profile may include, but are not limited to, completing tumor acquisition forms, submitting tumor acquisition forms for approval, approving tumor acquisition forms, printing tumor acquisition forms, and uploading tumor acquisition forms.

Users with a Patient Services profile may include, but are not limited to, Patient Services staff, Patient Advisors, hospital billing staff, hospital insurance managers, etc. Functions associated with the Patient Help Desk profile may include, but are not limited to, viewing Patient Help Desk cases, uploading files to cases, annotating cases, forwarding cases to a case manager, etc.

In some system embodiments, certain types of information are available for certain profiles. In some embodiments, information may be available to users with a certain profile only when performing certain types of functions, and not when performing other types of functions. For example, in some embodiments, COC/COI and manufacturing process information may be available through the Patient Help Desk profile to hospital billing managers and/or hospital insurance managers, but not visible to patient consultants. In some embodiments, production and COC/COI information may be visible, but not editable, by patient support personnel who perform functions such as scheduling patient treatment events, assisting the patient in the scheduling process, and/or assisting the patient in transporting to patient's treatment events.

For example, in some embodiments, a method of producing a cell therapy product by expanding a population of cells derived from a patient's tumor into a cell therapy product may include receiving the population of cells from the patient at a manufacturing facility based on a cell order request to produce the cell therapy product for the patient;

- 170 045580 generating, by the computing device, a patient-specific identifier including a cell order identifier associated with the cell order request;

initiation of the production process for a cell therapy product, including:

after receiving the population of cells at the manufacturing facility, scheduling, by the computing device, treatment events for the patient, initiating expansion of the cell therapy product from at least some population of cells using a cell expansion technique, and determining acceptance parameters of the expanded cell therapy product at a first time point and at a second time point time subsequent to the first time point, determining whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria associated with the corresponding time point, in response to determining that the acceptance parameters of the expanded cell therapy product meet the acceptance criteria at the first time point, continued expanding the cell therapy product from at least a portion of the resulting cell therapy product using a cell expansion technique up to a second time point, and in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at the second time point:

determining whether it is possible to re-exploit the cell therapy product using the cell expansion technique from a first time point based on the acceptability parameters at a second time point, in response to determining that re-execution is possible, re-performing the expansion of the cell therapy product from at least a portion of the cell therapy product therapy obtained at the second time point using the cell expansion technique from the first time point to obtain the cell therapy product, estimating the completion time of the cell therapy product expansion after re-executing the expansion of the cell therapy product from the first time point, and changing the treatment schedule using the computing device patient events and completion of subsequent expansion of the cell therapy product from the first time point, wherein rescheduling of patient treatment events is performed based on the estimated completion time of cell therapy product expansion and the timing of treatment events before or after infusion of the expanded cell therapy product into the patient, and provision of support services patients, such as travel support, health management, health insurance, reimbursement and other treatment-related services.

VI. Additional considerations.

The above examples are provided to provide those skilled in the art with complete disclosure and description of how to make and use embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors believe to be their invention. Modifications to the above-described methods of carrying out the invention, which are obvious to those skilled in the art, are included in the scope of the following claims. All patents and publications mentioned in the description indicate the level of qualification of specialists in the field of technology to which the invention relates.

All headings and section designations are used for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different headings and sections consistent with the spirit and scope of the invention described herein.

All references cited herein are hereby incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference. in its entirety for all purposes.

Many modifications and variations may be made according to this application without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited to the terms of the appended claims together with the full scope of equivalents covered by such claims.

Various examples of aspects of the invention are described in numbered paragraphs (1, 2, 3, etc.) for convenience. They are given as examples and do not limit the technology in question. The designations of the figures and reference numbers are given below as examples and for illustrative purposes only, and the items are not limited to such designations.

P.1. A method of producing a cell therapy product by expanding a population of cells derived from a patient's tumor into a cell therapy product, the method including receiving the population of cells from the patient at a manufacturing facility based on an order request for cells to produce a cell therapy product for the patient;

- 171 045580 generating, by the computing device, a patient-specific identifier including a cell order identifier associated with the cell order request;

initiating a process for manufacturing a cell therapy product including, after receiving a population of cells at a manufacturing facility, scheduling, by a computing device, patient treatment events, initiating propagation of the cell therapy product from at least some population of cells using a cell propagation technique, and determining acceptance parameters for the propagated cell therapy product at a first time point and at a second time point subsequent to the first time point, determining whether the expanded cell therapy product's acceptance parameters meet the acceptance criteria associated with the corresponding time point, in response to determining that the expanded cell therapy product's acceptance parameters meet acceptance criteria at the first time point, continuing to expand the cell therapy product from at least a portion of the resulting cell therapy product using a cell expansion technique until the second time point, and in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at the second moment of time:

determining whether it is possible to re-exploit the cell therapy product using the cell expansion technique from a first time point based on the acceptability parameters at a second time point, in response to determining that re-execution is possible, re-performing the expansion of the cell therapy product from at least a portion of the cell therapy product therapy obtained at the second time point using the cell expansion technique from the first time point to obtain the cell therapy product, estimating the completion time of the cell therapy product expansion after re-executing the expansion of the cell therapy product from the first time point, and changing the treatment schedule using the computing device patient events and completion of subsequent propagation of the cell therapy product from the first time point, wherein the change in the schedule of patient treatment events is performed based on the estimated time of completion of propagation of the cell therapy product and the time of treatment events before or after infusion of the propagated cell therapy product into the patient's body,

P.2. The method of claim 1, wherein the cell therapy product comprises T cells.

P.3. The method of claim 1, wherein the cell therapy product comprises tumor infiltrating lymphocytes (TILs).

P.4. The method of claim 1, wherein the patient treatment events include one or more of the patient's hospital stay, a date of resection, a date of lymphodepletion, a date of infusion of a cell therapy product into the patient, and a date of IL-2 treatment.

P.5. The method of claim 1, wherein determining whether the expanded cell therapy product's acceptance parameters meet the acceptance criteria comprises determining the expanded cell therapy product's acceptance parameters at a plurality of time points after the resulting cell therapy product has begun to propagate, the plurality of time points including a first and a second time point. moments in time.

P.6. The method of claim 5, wherein changing the patient's treatment event schedule includes changing the patient's treatment event schedule in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at any of the plurality of time points.

P.7. The method of claim 5, wherein rescheduling the patient's treatment events includes terminating the patient's treatment events in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at any of the plurality of time points due to contamination.

P.8. The method of claim 5, wherein in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at any of the plurality of time points due to contamination, subsequent expansion of the cell therapy product is stopped.

P.9. The method of claim 8, wherein determining the acceptability parameters of the expanded cell therapy product includes one or more of determining viability, sterility, cell count, mycoplasma count, CD3 count, endotoxin assay result, and Gram stain assay result.

P.10. The method of claim 9, wherein the cell expansion technique comprises a rapid expansion step, the method further comprising determining whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria prior to the rapid expansion step; and

- 172 045580 in response to determining that the expanded cell therapy product's eligibility parameters meet the eligibility criteria, setting a lymphodepletion date on a date prior to completion of production of the expanded cell therapy product, setting an infusion date on a date subsequent to completion of production of the expanded cell therapy product, and assigning an IL-2 treatment date after the infusion date.

P.11. The method according to claim 1, wherein the cell propagation technique includes culturing the cell therapy product in a separate bioreactor with a closed container.

P.12. The method of claim 1, wherein estimating the time to complete propagation of the cell therapy product after re-performing propagation of the cell therapy product from the first time point includes determining the time required to complete the propagation process from the first time point based on one or both of the acceptance parameters at the second time point. a point in time and acceptance parameters associated with the population of cells used to re-expand the cell therapy product from the first point in time.

P.13. The method of claim 1, wherein the cell order request for expansion of the cell therapy product is received from the hospital-side interface, the method further comprising transmitting, upon receipt of the cell order request, an acknowledgment including one or both of a patient-specific identifier and a cell order identifier , to the hospital-side interface that the cell order request associated with the patient has been accepted.

P.14. The method of claim 13, further comprising assigning a set of dates corresponding to a plurality of time points, including first and second time points, to determine whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria during expansion of the cell therapy product depending on the cell expansion technique and when the request to order cells is accepted.

P.15. The method of claim 13, further comprising transmitting, after changing the patient's treatment event schedule, to the hospital-side interface the updated patient treatment event schedule associated with the specific patient identifier.

P.16. The method of claim 15, further comprising transmitting to the logistics interface a receipt order associated with the patient-specific identifier based on the completion time; and transmitting to the hospital-side interface a schedule of patient treatment events associated with a patient-specific identifier based on completion time.

P.17. The method of claim 16, further comprising, in response to determining that re-expanding the cell therapy product from the first point in time is possible:

separating, by the computing device, the cell order identifier from the patient-specific identifier; and generating, by the computing device, a new cell order identifier associated with the cell order request, and associating the new cell order identifier with the patient-specific identifier.

P.18. The method of claim 17, wherein the cell order request for expansion of the cell therapy product is received from the hospital-side interface, the method further comprising transmitting a patient-specific identifier including a new cell order identifier and an estimated completion time for expansion of the cell therapy product to the interface on the hospital side.

P.19. The method of claim 17, further comprising generating, by the computing device, a new schedule of delivery and logistics events associated with the patient's treatment events based on the change in the patient's treatment event schedule, and transmitting the new schedule of delivery and logistics events associated with the patient-specific identifier , including a new cell order ID, into the logistics interface based on rescheduling of shipping and logistics events.

P.20. The method of claim 17, further comprising associating, by the computing device with the patient-specific identifier, at each point in time when a determination is made whether the eligibility parameters meet the defined eligibility criteria, a new cell order identifier including fields corresponding to each corresponding point in time, and the result of the determination.

P.21. The method of claim 20, wherein the scheduling includes scheduling, by the computing device, patient treatment events based on the patient-specific identifier including the new cell order identifier.

P.22. The method of claim 21, further comprising, in response to determining that re-execution is not possible, canceling, by the computing device, patient treatment events scheduled after the second time point.

P.23. The method of claim 22, further comprising transmitting information about the cancellation of the patient's treatment events to the hospital-side interface and the logistics interface.

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P.24. The method of claim 23, further comprising, in response to determining that re-execution is not possible, destroying the expanded cell therapy product and dissociating the patient-specific identifier from the cell order identifier.

P.25. A method of treating a patient with an expanded cell therapy product obtained by the method of claim 1, in accordance with the patient's modified schedule of treatment events.

P.26. A method of producing a cell therapy product for a patient, comprising receiving, at a computing device, a request for ordering cells to produce a cell therapy product for a patient, production slots at a plurality of manufacturing plants for producing the cell therapy product, wherein the production slots for a corresponding manufacturing plant are received at the computing device from the computer. a manufacturer subsystem associated with the respective manufacturing facility and a patient's advance treatment event schedule for treating the patient with the cell therapy product;

determining and displaying in a scheduling user interface, by the computing device, a plurality of available production slots for manufacturing the cell therapy product based on the patient's prior schedule of treatment events;

after selecting one of the available production slots, performing a procedure on the patient at the medical facility to obtain a solid tumor from the patient in accordance with the patient's preliminary treatment event schedule and the available production slot;

delivering the resulting solid tumor to a manufacturing facility corresponding to the available production window, in accordance with the available production window;

initiating, upon receipt of the resulting solid tumor at a manufacturing facility, the production of a cell therapy product from at least a portion of the resulting solid tumor using cell expansion techniques;

determining, during manufacture of the cell therapy product, the acceptance parameters of the manufactured cell therapy product at a first time point and at a second time point subsequent to the first time point, and whether the acceptance parameters meet the acceptance criteria associated with the relevant time point, wherein the acceptability parameters are determined based on the result analysis associated with the relevant point in time;

modifying, by the computing device, a production schedule for manufacturing the cell therapy product, production intervals appropriate to the manufacturing plant, and a preliminary schedule of patient treatment events based on whether the eligibility criteria are met at one or both of the first and second time points; and completion of production of the cell therapy product in accordance with the modified production schedule if acceptance criteria are met at both the first and second time points.

P.27. The method of claim 26, further comprising delivering the produced cell therapy product to a medical facility.

P.28. The method of claim 26, further comprising, upon receiving the cell order request, generating, by the computing device, a patient-specific identifier associated with the patient and the cell order request.

P.29. The method of claim 28, further comprising automatically generating, by the computing device, a shipping label for the resulting solid tumor container, a shipping label including a patient-specific identifier, a cell order request, a production slot, and a manufacturing facility corresponding to the available production slot.

P.30. The method of claim 29, further comprising automatically generating, by the computing device, a manufacturing label for a container for the resulting solid tumor, wherein the manufacturing label comprises a point in time corresponding to the manufacturing process used to manufacture the cell therapy product when the manufacturing label is generated, a bar code identifying the container , patient-specific identifier, manufacturing steps completed at a given time, acceptance parameters associated with completed processes, and the manufacturing process being performed at a given time.

P.31. The method of claim 26, wherein the cell therapy product comprises T cells.

P.32. The method of claim 26, wherein the cell therapy product comprises tumor infiltrating lymphocytes (TILs).

P.33. The method of claim 26, wherein the patient treatment events include one or more of the patient's hospital stay, a date of resection, a date of lymphodepletion, a date of infusion of a cell therapy product into the patient, and a date of IL-2 treatment.

P.34. The method of claim 26, wherein determining whether the acceptance parameters of the manufactured cell therapy product meet the acceptance criteria comprises determining the acceptance parameters of the manufactured cell therapy product at a plurality of time points after

- 174 045580 the start of propagation of the received cell therapy product, the plurality of time points including first and second time points.

P.35. The method of claim 34, further comprising rescheduling, by the computing device, the patient's treatment events and completing subsequent propagation of the cell therapy product, wherein rescheduling the patient's treatment events includes rescheduling the patient's treatment events in response to determining that the acceptance parameters of the produced cell therapy product therapies do not meet eligibility criteria at any of multiple time points.

P.36. The method of claim 34, wherein rescheduling the patient's treatment events includes terminating the patient's treatment events in response to determining that the acceptance parameters of the manufactured cell therapy product do not meet the acceptance criteria at any of the plurality of time points due to contamination.

P.37. The method of claim 34, wherein in response to determining that the acceptance parameters of the produced cell therapy product do not meet the acceptance criteria at any of the plurality of time points due to contamination, subsequent propagation of the cell therapy product is stopped.

P.38. The method of claim 37, wherein determining the acceptance parameters of the manufactured cell therapy product includes one or more of determining viability, sterility, cell count, mycoplasma count, CD3 count, endotoxin assay result, and Gram stain assay result.

P.39. The method of claim 38, wherein the cell expansion technique comprises a rapid expansion step, the method further comprising determining whether the acceptance parameters of the produced cell therapy product meet the acceptance criteria prior to the rapid expansion step; and in response to determining that the acceptance parameters of the manufactured cell therapy product meet the eligibility criteria, setting a lymphodepletion date on a date prior to completion of production of the cell therapy product, setting an infusion date on a date subsequent to completion of production of the cell therapy product, and setting a treatment date IL-2 after the infusion date.

P.40. The method of claim 26, wherein the cell propagation technique comprises culturing the cell therapy product in a separate bioreactor with a closed container.

P.41. The method of claim 26, further comprising estimating, by means of a computing device, a time to complete propagation of the cell therapy product, wherein estimating the time to complete propagation of the cell therapy product after re-executing propagation of the cell therapy product from the first point in time includes determining the time required to complete the propagation process from the first point in time, based on one or both of the acceptance parameters at the second time point and the acceptance parameters associated with the population of cells used to re-expand the cell therapy product from the first time point.

P.42. The method of claim 29, wherein the cell order request for expansion of the cell therapy product is received from the hospital-side interface, the method further comprising transmitting, upon receipt of the cell order request, a confirmation including one or both of a patient-specific identifier and a cell order identifier , to the hospital-side interface that the cell order request associated with the patient has been accepted.

P.43. The method of claim 42, further comprising assigning a set of dates corresponding to a plurality of time points, including the first and second time points, to determine whether the acceptance parameters of the manufactured cell therapy product meet the acceptance criteria during propagation of the cell therapy product depending on the cell propagation technique and when the cell order request is accepted.

P.44. The method of claim 43, further comprising transmitting, after changing the patient's treatment event schedule, to the hospital-side interface the updated patient treatment event schedule associated with the specific patient identifier.

P.45. The method of claim 44, further comprising transmitting to the logistics interface a receipt order associated with the patient-specific identifier based on the completion time; and transmitting to the hospital-side interface a schedule of patient treatment events associated with a patient-specific identifier based on completion time.

P.46. The method of claim 45, further comprising, in response to determining that re-expanding the cell therapy product from the first point in time, allowing the computing device to separate the cell order identifier from the patient-specific identifier, and allowing the computing device to generate a new order identifier cells associated with the cell order request, and associating the new cell order ID with the patient-specific ID.

P.47. The method of claim 46, wherein the cell order request for expansion of the cell therapy product is received from the hospital-side interface, the method further comprising transmitting a patient-specific identifier including a new cell order identifier and an estimated completion time for expansion of the cell therapy product to the interface on the hospital side.

P.48. The method of claim 47, further comprising generating, by the computing device, a new schedule of delivery and logistics events associated with the patient's treatment events based on the change in the patient's treatment event schedule, and transmitting the new schedule of delivery and logistics events associated with the patient-specific identifier , including a new cell order ID, into the logistics interface based on rescheduling of shipping and logistics events.

P.49. The method of claim 48, further comprising associating, by the computing device with the patient-specific identifier, at each point in time when a determination is made whether the eligibility parameters meet the eligibility criteria, a new cell order identifier including fields corresponding to each corresponding point in time, and result of the determination.

P.50. The method of claim 49, wherein the scheduling includes scheduling, by the computing device, patient treatment events based on the patient-specific identifier including the new cell order identifier.

P.51. The method of claim 50, further comprising, in response to determining that re-execution is not possible, canceling, by the computing device, patient treatment events scheduled after the second time point.

P.52. The method of claim 51, further comprising transmitting information about the cancellation of the patient's treatment events to the hospital-side interface and the logistics interface.

P.53. The method of claim 52, further comprising, in response to determining that re-execution is not possible, destroying the expanded cell therapy product and dissociating the patient-specific identifier from the cell order identifier.

P.54. A method of treating a patient with a manufactured cell therapy product obtained by the method of claim 26 in accordance with the patient's modified schedule of treatment events.

P.55. A method of producing a cell therapy product, comprising receiving into a manufacturing facility a solid tumor obtained from a patient;

generating, by a computing device, a manufacturing label for a manufacturing container for use in a process for manufacturing a cell therapy product from at least a portion of the resulting solid tumor using a cell expansion technique, wherein the manufacturing label includes information related to the patient, the manufacturing process, and the quality of the product produced cell therapy;

initiating a process for manufacturing a cell therapy product, comprising performing a procedure on a patient in a medical facility to obtain a solid tumor from the patient, delivering the resulting solid tumor to the manufacturing facility, upon receipt of the resulting solid tumor at the manufacturing facility, dynamically scheduling, using a computing device, treatment events for the patient, wherein dynamically scheduling depends on the acceptance parameters of the subsequently obtained expanded cell therapy product, initiating expansion of the cell therapy product from at least a portion of the resulting solid tumor using a cell expansion technique and determining the acceptance parameters of the expanded cell therapy product at multiple points in time, performing quality control to determine the parameters acceptability of the manufactured cell therapy product at multiple points in time;

receiving in the computing device the acceptability parameters of the produced cell therapy product;

generating, by the computing device, an updated manufacturing label corresponding to each of the plurality of time points, wherein the updated manufacturing label includes updated information associated with the quality of the cell therapy product manufactured, wherein the updated information includes acceptance parameters at the corresponding point in time;

reading, by the computing device, the updated production label at each of a plurality of times; and completing propagation of the cell therapy product based on information read from the updated manufacturing label at each of a plurality of time points.

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P.56. The method of claim 55, further comprising providing, by the computing device, a warning signal if the patient information on the updated manufacturing label for the subsequent manufacturing step does not match the patient information on the manufacturing label for the immediately preceding manufacturing step, or the acceptability parameters on the updated The manufacturing label for a given point in time in the manufacturing process does not meet the acceptance criteria for that point in time in the manufacturing process, where the acceptance parameters include one or more of viability, sterility, cell count, mycoplasma count, CD3 count, endotoxin test result, and Gram stain test result.

P.57. The method of claim 55, wherein the patient-related information comprises a patient-specific identifier and a cell order identifier associated with a request to order cells to manufacture a cell therapy product for the patient.

P.58. The method of claim 55, wherein the cell propagation technique comprises culturing the cell therapy product in a separate bioreactor with a closed container.

P.59. The method of claim 55, wherein the manufacturing label includes a bar code encoding information related to the patient, the manufacturing process, and the quality of the cell therapy product produced.

P.60. The method of claim 56, further comprising assigning a set of dates corresponding to a plurality of time points, including a first time point and a second time point subsequent to the first time point, to determine whether the acceptance parameters of the manufactured cell therapy product meet acceptance criteria during the manufacturing process depending on the cell propagation technique used and when the cell order request is accepted by the manufacturing facility.

P.61. The method of claim 60, further comprising integrating with a logistics interface, receiving courier status information through the courier computing subsystem, wherein the courier status information includes, and in response to receiving the courier status information, determining a delivery schedule for delivery of the manufactured cell therapy product based on the determined manufacturing schedules and generating a shipping label for the shipping container containing the manufactured cell therapy product.

P.62. The method of claim 60, further comprising transmitting to the logistics entity a delivery request based on a determined delivery schedule.

P.63. The method of claim 60, further comprising generating a shipping label for a shipping container containing the manufactured cell therapy product prior to performing final quality control, wherein the shipping label indicates that the manufactured cell therapy product is not to be released unless the result of final quality control indicates, that the relevant acceptance parameters meet the relevant acceptance criteria.

P.64. The method of claim 56, further comprising, after determining that the acceptance parameters of the manufactured cell therapy product meet the acceptance criteria, determining a completion date for production of the cell therapy product;

generating, by the computing device, a schedule of patient treatment events corresponding to the use of the cell therapy product to treat the patient based on the completion date;

transmission to the logistics interface of the receiving order based on the completion date; as well as transferring the patient’s treatment event schedule to the interface on the hospital side.

P.65. The method of claim 55, wherein the cell therapy product comprises T cells.

P.66. The method of claim 55, wherein the cell therapy product comprises tumor infiltrating lymphocytes (TILs).

P.67. The method of claim 55, wherein the patient treatment events include one or more of the patient's hospital stay, a date of resection, a date of lymphodepletion, a date of infusion of a cell therapy product into the patient, and a date of IL-2 treatment.

P.68. The method of claim 56, wherein determining whether the expanded cell therapy product's acceptance parameters meet the acceptance criteria comprises determining the expanded cell therapy product's acceptance parameters at a plurality of time points after initiation of expansion of the resulting cell therapy product, the plurality of time points including a first and a second time point. moments in time.

P.69. The method of claim 68, further comprising rescheduling, by the computing device, the patient's treatment events and completing subsequent propagation of the cell therapy product, wherein rescheduling the patient's treatment events includes changing

- 177 045580 patient treatment event schedules in response to a determination that the eligibility parameters of the expanded cell therapy product do not meet the eligibility criteria at any of a plurality of time points.

P.70. The method of claim 68, wherein rescheduling the patient's treatment events includes terminating the patient's treatment events in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at any of the plurality of time points due to contamination.

P.71. The method of claim 68, wherein in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at any of the plurality of time points due to contamination, subsequent expansion of the cell therapy product is stopped.

P.72. The method of claim 71, wherein determining the acceptance parameters of the expanded cell therapy product includes one or more of determining viability, sterility, cell count, mycoplasma count, CD3 count, endotoxin assay result, and Gram stain assay result.

P.73. The method of claim 72, wherein the cell expansion technique comprises a rapid expansion step, the method further comprising determining whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria prior to the rapid expansion step; and in response to determining that the expanded cell therapy product's eligibility parameters meet the eligibility criteria, setting a lymphodepletion date on a date prior to completion of manufacturing of the cell therapy product, setting an infusion date on a date subsequent to completion of manufacturing of the cell therapy product, and setting a treatment date IL-2 after the infusion date.

P.74. The method of claim 55, wherein the cell propagation technique comprises culturing the cell therapy product in a separate bioreactor with a closed container.

P.75. The method of claim 55, wherein estimating the time to complete propagation of the cell therapy product after re-performing propagation of the cell therapy product from the first time point includes determining the time required to complete the propagation process from the first time point based on one or both of the acceptance parameters at the second time point. a point in time and acceptance parameters associated with the population of cells used to re-expand the cell therapy product from the first point in time.

P.76. The method of claim 57, wherein the cell order request for expansion of the cell therapy product is received from the hospital-side interface, the method further comprising transmitting, upon receipt of the cell order request, a confirmation including one or both of a patient-specific identifier and a cell order identifier , to the hospital-side interface that the cell order request associated with the patient has been accepted.

P.77. The method of claim 76, further comprising assigning a set of dates corresponding to a plurality of time points, including first and second time points, to determine whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria during expansion of the cell therapy product depending on the cell expansion technique and when the request to order cells is accepted.

P.78. The method of claim 76, further comprising transmitting, after changing the patient's treatment event schedule, to the hospital-side interface the updated patient treatment event schedule associated with the specific patient identifier.

P.79. The method of claim 78, further comprising transmitting to the logistics interface a receipt order associated with the patient-specific identifier based on the completion time; and transmitting to the hospital-side interface a schedule of patient treatment events associated with the patient-specific identifier based on the completion time.

P.80. The method of claim 79, further comprising, in response to determining that re-expanding the cell therapy product from the first point in time, allowing the computing device to separate the cell order identifier from the patient-specific identifier, and allowing the computing device to generate a new order identifier cells associated with the cell order request, and associating the new cell order ID with the patient-specific ID.

P.81. The method of claim 80, wherein the cell order request for expansion of the cell therapy product is received from the hospital-side interface, the method further comprising transmitting a patient-specific identifier including a new cell order identifier and an estimated completion time for expansion of the cell therapy product to the interface on the hospital side.

- 178 045580

P.82. The method of claim 80, further comprising generating, by the computing device, a new schedule of delivery and logistics events associated with the patient's treatment events based on the change in the patient's treatment event schedule, and transmitting the new schedule of delivery and logistics events associated with the patient-specific identifier , including a new cell order ID, into the logistics interface based on rescheduling of shipping and logistics events.

P.83. The method of claim 80, further comprising associating, by the computing device with the patient-specific identifier, at each point in time when a determination is made whether the eligibility parameters meet the defined eligibility criteria, a new cell order identifier including fields corresponding to each corresponding point in time, and the result of the determination.

P.84. The method of claim 83, wherein the scheduling includes scheduling, by the computing device, patient treatment events based on the patient-specific identifier including the new cell order identifier.

P.85. The method of claim 84, further comprising, in response to determining that re-execution is not possible, canceling, by the computing device, patient treatment events scheduled after the second time point.

P.86. The method of claim 85, further comprising transmitting information about the cancellation of the patient's treatment events to the hospital-side interface and the logistics interface.

P.87. The method of claim 86, further comprising, in response to determining that re-execution is not possible, destroying the expanded cell therapy product and dissociating the patient-specific identifier from the cell order identifier.

P.88. A method of treating a patient with an expanded cell therapy product obtained by the method of claim 55 in accordance with the patient's modified schedule of treatment events.

Claims (30)

1. A method for coordinating the production of expanded T cells for treating cancer in a patient, the method comprising producing a cell therapy product by expanding a population of cells derived from the patient's tumor into a cell therapy product, the production comprising receiving, by a computing device, a request to order cells for propagating a population of cells for the patient; generating, by the computing device, a cell order identifier including an individual patient identifier associated with the cell order request; initiation of the cell therapy product manufacturing process; after receiving a population of cells at a manufacturing facility, determining, using a computing device, a preliminary schedule for the cell expansion process and a preliminary schedule for the patient's treatment events, initiating expansion of the cell therapy product from at least a portion of the resulting cell population using the cell expansion process, and determining acceptance parameters for the expanded cell product therapy at multiple points in time during the cell expansion process, determining whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria, and in response to determining that the acceptance parameters of the expanded cell therapy product meet the acceptance criteria, completing the cell expansion process in accordance with the preliminary schedule for said process to produce a manufactured cell therapy product, and treating the patient in accordance with the patient's preliminary treatment event schedule, wherein the patient's treatment events include one or more of the patient's hospital stay, a date of resection, a date of lymphodepletion, a date of infusion into the patient of the manufactured cell therapy product therapy and dates of IL-2 treatment. 2. The method of claim 1, further comprising, in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria, determining, by the computing device, a modified process schedule and a modified patient treatment event schedule, completing subsequent expansion of the cell therapy product at in accordance with the modified process schedule, wherein the modified patient treatment event schedule is based on the clinically determined time interval between the infusion of the manufactured cell therapy product into the patient's body and the patient treatment events before or after the infusion of the manufactured cell therapy product into the patient's body. 3. The method of claim 2, wherein the plurality of time points during the cell expansion process include a first time point and a second time point subsequent to the first time point, the method further comprising determining whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria at the first time point, in response to determining that the acceptance parameters of the expanded cell therapy product meet the acceptance criteria at the first time point, continuing to expand the cell therapy product in accordance with the preliminary process schedule. 4. The method of claim 3, further comprising, in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at the second time point, determining whether the expansion of the cell therapy product can be repeated using the cell expansion process from the first time point based on acceptance parameters at a second time point, in response to determining that re-execution is possible, re-expanding the cell therapy product from at least a portion of the cell therapy product obtained at the second time point using the cell expansion process from the first time point to obtain of the cell therapy product, estimating the completion time of propagation of the cell therapy product after re-performing propagation of the cell therapy product from the first time point, and determining, using the computing device, a modified schedule of treatment events for the patient, and completing subsequent propagation of the cell therapy product from the first time point, wherein the modified schedule of treatment events patient is based on the estimated time of completion of cell therapy product propagation and the timing of treatment events before or after infusion of the produced cell therapy product into the patient's body. 5. The method of claim 4, wherein in response to determining that the acceptance parameters of the expanded cell therapy product do not meet the acceptance criteria at any of the plurality of time points due to contamination, subsequent expansion of the cell therapy product is stopped. 6. The method of claim 1, wherein determining the acceptance parameters of the expanded cell therapy product includes one or more of determining viability, sterility, cell count, mycoplasma count, CD3 count, endotoxin assay result, and Gram stain assay result. 7. The method of claim 1, wherein the cell expansion process comprises a rapid expansion step, the method further comprising determining whether the acceptance parameters of the expanded cell therapy product meet the acceptance criteria prior to the rapid expansion step; and in response to determining that the expanded cell therapy product's eligibility parameters meet the eligibility criteria, setting a lymphodepletion date on a date prior to completion of production of the expanded cell therapy product, setting an infusion date on a date subsequent to completion of production of the expanded cell therapy product, and assigning IL-2 treatment dates after the infusion date. 8. A method of producing a cell therapy product for treating cancer in a patient, comprising receiving, at a computing device, a request to order cells to produce a cell therapy product for the patient, information about production facilities at a plurality of manufacturing facilities for producing the cell therapy product, wherein information about the production facilities for the respective manufacturing facility is received at the computing device from a manufacturer's computer subsystem associated with the respective manufacturing facility and the patient's preliminary schedule of treatment events for treating the patient with the cell therapy product; determining and displaying in a scheduling user interface, by the computing device, a plurality of available production facilities for manufacturing the cell therapy product based on the patient's prior treatment event schedule; after selecting one of the available manufacturing facilities, performing a procedure on the patient at the medical facility to obtain a solid tumor from the patient in accordance with the patient's preliminary schedule of treatment events and the available manufacturing facilities; delivering the resulting solid tumor or a first population of cells derived from the resulting solid tumor to a manufacturing facility corresponding to available production capacity, with appropriate available production capacity; updating the availability of production capacity for the production of a cell therapy product based on a preliminary schedule for the production process of a cell therapy product; in a manufacturing facility, obtaining a second population of cells from the resulting solid tumor and initiating a process for producing a cell therapy product from some of the cells of the first population or cells of the second population; determining, during manufacture of the cell therapy product, acceptability parameters of the manufactured cell therapy product and whether the acceptance parameters meet the acceptance criteria at a plurality of time points, including a first time point and a second time point subsequent to the first time point, wherein the acceptability parameters are determined based on the result of the analysis; completion, in response to a determination that the acceptance criteria have been met, of production of the cell therapy product. 9. The method of claim 8, further comprising, upon receipt at the manufacturing facility of the resulting solid tumor or a first population of cells derived from the resulting solid tumor, generating, by the computing device, an updated manufacturing label for a manufacturing container for use in the manufacturing process of the cell therapy product from at least a portion of the resulting solid tumor or first population of cells through a cell expansion process, wherein the manufacturing label includes information related to the patient, the manufacturing process of the cell therapy product, and the quality of the cell therapy product produced; generating, by the computing device, an updated manufacturing label corresponding to each of the plurality of time points, wherein the updated manufacturing label includes updated information related to the quality of the manufactured cell therapy product containing acceptance parameters at the corresponding point in time; reading, by the computing device, the updated production label at each of the plurality of times; and completing propagation of the cell therapy product based on the information read from the updated production label at each of the plurality of times. 10. The method of claim 9, further comprising modifying, by the computing device, a pre-schedule of the cell therapy product manufacturing process, the availability of manufacturing facilities associated with the manufacturing facility, and a pre-schedule of patient treatment events based on whether acceptance criteria are met at multiple points in time, including one or both of the first and second time points to obtain a modified production schedule and a modified patient treatment event schedule; and completing production of the cell therapy product in accordance with the modified production schedule, wherein determining whether the acceptance parameters for the manufactured cell therapy product meet the acceptance criteria further includes determining, during production of the cell therapy product, the acceptance parameters of the manufactured cell therapy product at the first and second time points. , and whether the acceptability parameters meet the acceptance criteria associated with the relevant point in time, wherein the acceptability parameters are determined based on the result of the analysis associated with the relevant point in time; the method further includes completing production of the cell therapy product in accordance with the modified production schedule if the acceptance criteria are not met at one or both of the first and second time points. 11. The method of claim 10, further comprising generating, by the computing device, a new schedule for a new schedule of delivery and logistics events associated with the patient's treatment events based on the modified schedule of patient's treatment events, and transmitting the new schedule of delivery and logistics events associated with with a patient-specific ID, including a new cell order ID, into the logistics interface. 12. The method of claim 9, wherein the manufacturing label includes a bar code encoding information related to the patient, the manufacturing process, and the quality of the cell therapy product produced. 13. The method of claim 8, further comprising assigning a set of dates corresponding to a plurality of time points, including first and second time points, to determine whether the acceptance parameters of the manufactured cell therapy product meet acceptance criteria during the cell therapy product manufacturing process depending on cell propagation techniques and when the request for ordering cells is accepted. 14. The method of claim 8, wherein the acceptance parameters include one or more of viability, sterility, cell count, mycoplasma count, CD3 count, enzyme assay result - 181 045580 dotoxin and Gram stain analysis results. 15. The method of claim 8, further comprising automatically generating, by the computing device, a shipping label for a container for the resulting solid tumor or a first population of cells derived from the resulting solid tumor, a shipping label including an individual patient identifier, a cell order request, available manufacturing facilities facilities and manufacturing facility with associated production capacity available; automatically generating, by a computing device, a manufacturing label for a container for the resulting solid tumor or a first population of cells derived from the resulting solid tumor, wherein the manufacturing label comprises a point in time corresponding to the manufacturing process used to manufacture the cell therapy product when the manufacturing label is generated, a code, identifying container, individual patient identifier, manufacturing steps completed at a given time, acceptance parameters associated with completed processes, and manufacturing process being performed at a given time; and providing, by the computing device, an alert if the patient information on the updated manufacturing label for the subsequent manufacturing step does not match the patient information on the manufacturing label for the immediately preceding manufacturing step, or the acceptability parameters on the updated manufacturing label for that point in time of the manufacturing step. processes do not meet the acceptance criteria for that point in time in the production process. 16. The method of claim 8, wherein the process for manufacturing the cell therapy product includes a rapid expansion step, and the method further includes determining whether the acceptance parameters of the manufactured cell therapy product meet the acceptance criteria prior to the rapid expansion step; and in response to determining that the acceptance parameters of the manufactured cell therapy product meet the eligibility criteria, setting a lymphodepletion date on a date prior to completion of production of the cell therapy product, setting an infusion date on a date subsequent to completion of production of the cell therapy product, and setting a treatment date IL-2 after the infusion date. 17. The method of claim 8, wherein the cell contains T cells. 18. The method of claim 17, wherein the cell therapy product comprises tumor infiltrating lymphocytes (TILs). 19. The method of claim 8, further comprising integrating the computing device with the logistics interface, receiving courier status information through the courier computing subsystem, wherein the courier status information includes, in response to receiving the courier status information, determining a delivery schedule for delivery of the manufactured product cell therapy product based on a preliminary manufacturing process schedule for the cell therapy product, and generating a shipping label for a shipping container containing the manufactured cell therapy product, transmitting a shipping request to the logistics facility based on the determined shipping schedule, and generating a shipping label for the shipping container containing the manufactured cell therapy product, until final quality control is performed, with the shipping label indicating that the cell therapy product produced is not to be released unless the result of the final quality control shows that the relevant acceptance parameters are met. 20. The method of claim 8, further comprising determining an estimated completion date for production of the cell therapy product based on a preliminary schedule for the production process of the cell therapy product; transmitting to the logistics interface an order based on the completion date; and transmitting to the hospital-side interface a preliminary schedule of patient treatment events. 21. The method of claim 10, further comprising determining a modified production completion date for the cell therapy product based on the modified production schedule; transmitting to the logistics interface the receipt order associated with the individual patient ID based on the modified completion date; and transmitting to the hospital-side interface a schedule of patient treatment events associated with the individual patient ID based on the modified completion date. 22. A method of producing a cell therapy product, comprising receiving into a manufacturing facility a solid tumor obtained from a patient, or a first population of cells derived from a solid tumor; generating, by the computing device, a manufacturing label for a manufacturing container for use in the manufacture of a cell therapy product from at least a portion of the resulting solid tumor or first population of cells, wherein the manufacturing label includes information associated with a patient, a process for manufacturing the cell therapy product therapy and the quality of the cell therapy product produced; initiating a process for manufacturing a cell therapy product, including performing a procedure on a patient in a medical facility to obtain a solid tumor from the patient or a first population of cells derived from a solid tumor, delivering the resulting solid tumor or first population of cells to a manufacturing facility, after receiving the resulting solid tumor at the manufacturing facility enterprise, dynamically scheduling, by means of a computing device, treatment events for a patient, wherein the dynamic scheduling is dependent on the acceptability parameters of a subsequently produced cell therapy product, initiating propagation of the cell therapy product from at least a portion of the resulting solid tumor or first cell population using a cell propagation technique, and determining parameters of propagation acceptance of the cell therapy product at multiple points in time, performing quality control to determine parameters of acceptability of the produced cell therapy product at multiple points in time; receiving in the computing device the acceptability parameters of the produced cell therapy product; generating, by the computing device, an updated manufacturing label corresponding to each of the plurality of time points, wherein the updated manufacturing label includes updated information associated with the quality of the cell therapy product manufactured, wherein the updated information includes acceptance parameters at the corresponding point in time; reading, by the computing device, the updated production label at each of a plurality of times; and completing propagation of the cell therapy product based on information read from the updated manufacturing label at each of a plurality of time points. 23. The method of claim 22, further comprising providing, by the computing device, an alert if the patient information on the updated manufacturing label for the subsequent manufacturing step does not match the patient information on the manufacturing label for the immediately preceding manufacturing step or the acceptance parameters at the updated manufacturing label for a given point in time of the manufacturing process does not meet the acceptance criteria for that point in time of the manufacturing process, where the acceptance parameters include one or more of viability, sterility, cell count, mycoplasma count, CD3 count, endotoxin test result and Gram stain test result . 24. The method of claim 22, wherein the patient-related information comprises a patient-specific identifier and a cell order identifier associated with a request to order cells to manufacture a cell therapy product for the patient. 25. The method of claim 22, wherein the cell propagation technique comprises culturing the cell therapy product in a separate bioreactor with a closed container. 26. The method of claim 22, wherein the manufacturing label includes a bar code encoding information related to the patient, the manufacturing process of the cell therapy product, and the quality of the cell therapy product produced. 27. The method of claim 22, further comprising assigning a set of dates corresponding to a plurality of time points, including a first time point and a second time point subsequent to the first time point, to determine whether the acceptance parameters of the manufactured cell therapy product meet the acceptance criteria at the time process for manufacturing a cell therapy product depending on the cell propagation technique used and when the cell order request is accepted by the manufacturing facility. 28. The method of claim 27, further comprising receiving courier status information through the courier computing subsystem, in response to receiving the courier status information, determining a delivery schedule for delivery of the manufactured cell therapy product based on the determined production schedule, and generating a shipping label for the shipping container. containing a manufactured cell therapy product. 29. The method of claim 27, further comprising transmitting to the logistics enterprise a delivery request based on a determined delivery schedule. - 183 045580 30. The method of claim 22, further comprising, after determining that the acceptance parameters of the manufactured cell therapy product meet the acceptance criteria, determining a completion date for production of the cell therapy product; generating, by the computing device, a schedule of patient treatment events corresponding to the use of the cell therapy product to treat the patient based on the completion date; transmission to the logistics interface of the receiving order based on the completion date; as well as transferring the patient’s treatment event schedule to the interface on the hospital side.