CN118043067A

CN118043067A - Chimeric TIM4 receptors and uses thereof

Info

Publication number: CN118043067A
Application number: CN202280065559.5A
Authority: CN
Inventors: D·M·科瑞; B·齐涅维奇; S·托马斯
Original assignee: Cero Therapeutics Inc
Current assignee: Cero Therapeutics Holdings Inc
Priority date: 2021-07-28
Filing date: 2022-07-28
Publication date: 2024-05-14

Abstract

The present disclosure relates to chimeric Tim4 receptors, host cells modified to comprise chimeric Tim4 receptor molecules, and methods of making and using such receptor molecules and modified cells.

Description

Chimeric TIM4 receptors and uses thereof

Reference to an electronic sequence Listing

The contents of the electronic sequence listing (200265_4180wo_sequential listing. Xml; size: 266,847 bytes; and date of creation: 2022, 7, 28 days) are incorporated herein by reference in their entirety.

Background

Upon exposure to antigen, the primary antigen-specific T cells undergo activation, which promotes their clonal expansion, differentiation, and development into functional effector T cells that can kill cells expressing the cognate antigen (e.g., tumor cells). After antigen clearance, most effector T cells undergo apoptosis, and a subset of surviving effector T cells differentiate into memory T cells, which can provide long-term protection against antigen re-exposure. However, prolonged antigen exposure may lead to T cell depletion, enabling tumor cell survival. T cell depletion refers to a dysfunctional state obtained by T cells undergoing sustained TCR stimulation, characterized by upregulation of immune checkpoint molecules (e.g., PD-1, CTLA-4, tim-3) expression, impaired effector function, dysproliferation, and metabolic defects. Depletion of engineered T cells expressing Chimeric Antigen Receptors (CARs) may also occur.

Disclosure of Invention

In one aspect, the present disclosure provides a chimeric Tim4 receptor comprising a single-chain chimeric protein comprising: (a) an extracellular domain comprising a Tim4 binding domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain; and (c) a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. In some embodiments, the extracellular domain of a chimeric Tim4 receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain. In some embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17.

In some embodiments, the chimeric Tim4 receptor comprises: (a) An extracellular domain comprising a Tim4 binding domain, said Tim4 binding domain comprising the amino acid sequence of SEQ ID No. 6; (b) An intracellular signaling domain, wherein the intracellular signaling domain comprises: (i) A primary CD28 intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 12, a secondary TLR2TIR intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 17, and a tertiary CD3 zeta intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 14; or (ii) a primary CD28 intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 12, a secondary CD3 ζ intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 14, and a tertiary TLR2TIR intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 17; and (c) a CD28 transmembrane domain comprising the amino acid sequence of SEQ ID NO. 11, which is located between and connects the extracellular domain and the CD28 intracellular signaling domain.

In some embodiments, the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID NO:18 or SEQ ID NO:18 lacking amino acids 1-24.

In some embodiments, the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID NO. 19 or SEQ ID NO. 19 lacking amino acids 1-24.

Also provided herein are polynucleotides encoding the chimeric Tim4 receptors of the disclosure, vectors comprising the polynucleotides encoding the chimeric Tim4 receptors, and host cells expressing the chimeric Tim4 receptors.

Also provided herein are methods of using polynucleotides encoding chimeric Tim4 receptors, chimeric Tim4 receptor vectors, or host cells expressing chimeric Tim4 receptors in methods of treating a subject, optionally in combination with additional therapeutic agents. In some embodiments, the methods are for treating cancer.

Drawings

FIGS. 1A-1D: in vitro co-culture systems for assessing T cell antigen presenting function showed that the addition of T cells containing chimeric Tim4 receptors with TLR intracellular signaling domains added to the CD3 zeta signaling domain (and optionally the CD28 signaling domain) showed enhanced ability to act as Antigen Presenting Cells (APCs). Fig. 1A: pCTX247 (also referred to herein as CER 247) and pCTX1107 (also referred to herein as CER1107 or CER 1236) chimeric Tim4 receptor and pCTX 1107T cells, these pCTX 1107T cells were pulsed with E7 peptide and co-cultured with E7 specific T cells to assess their antigen presenting capacity. Fig. 1B: the E7-specific proliferative response was measured by CT Violet dye dilution after 6 days in the presence of autologous CER-T (CER with or without TLR) pulsed with E7 peptide. Fig. 1C: the addition of TLR-2ICD (pCTX, 1107) triggered the proliferative response of E7-specific TCRs, whereas CER without TLR was less irritating. All data were collected by FACS. Cell tracking using anti-mouse TCRb showed E7TCR-T cells. During co-culture, E7TCR-T cells were labeled with CT Violet. Fig. 1D: the percentage of E7TCR cells proliferated in culture in live CD3+ cells as determined by flow cytometry based on staining of CELL TRACE violet low E7 TCRb+ cells.

Fig. 2: t cells containing a chimeric Tim4 receptor with a TLR intracellular signaling domain added to a CD3 zeta signaling domain (and optionally a CD28 signaling domain) are strong stimulatory factors of an autologous E7-specific T cell response. CER-T cells were pulsed with E7 peptide and tested for their ability to trigger an autologous E7-specific T cell response. CD25 (a T cell activation marker) was assessed on E7 TCR-T cells 24 hours after the CER-T cells were pulsed with E7 peptide. CER-T pCTX1107 contains the TLR-2 intracellular domain (ICD) and is a strong stimulatory factor for E7-specific activation.

Fig. 3: t cells containing a chimeric Tim4 receptor with a TLR intracellular signaling domain added to a CD3 zeta signaling domain (and optionally a CD28 signaling domain) are strong stimulatory factors of an autologous E7-specific T cell response. CER-T cells were pulsed with E7 peptide and tested for their ability to trigger an autologous E7-specific T cell response. 24 hours after the CER-T cells were pulsed with E7 peptide, CD69 (an early marker for T cell activation) was assessed on E7 TCR-T cells. CER-T pCTX1107 contains the TLR-2 intracellular domain and is a strong stimulatory factor for E7-specific activation.

Fig. 4: robust cell surface chimeric Tim4 receptor expression and detection using anti-Tim 4 antibodies. Chimeric Tim4 receptor cell surface staining was assessed on day 5 post transduction using an anti-Tim 4 antibody (9F 4). pCTX1107 contains the TLR-2 intracellular sequence. The lentiviral cassette contains a p2A fragment followed by a truncated EGFRt polypeptide.

Fig. 5A-5C: pCTX131 CER-T cells enhance the efficacy of CD1928z CAR-T cells. Fig. 5A: ptd-Ser induced kinetics on JeKo-1 MCL cells in response to CD1928z CAR-T cells. JeKo-1 MCL cells were co-cultured at increased effector: target ratio and Ptd-Ser induction was assessed over time. The kinetics plot shows the percentage of viable JeKo-1 target that binds to rTim-4, a Ptd-Ser binding protein. Top of fig. 5B: jeKo-1 cells were co-cultured with pCTX184 (1928 z) + pCTX131, pCTX184+CTX156 control T cells or pCTX184 cells alone at a ratio of 1:1T cells to JeKo-1 for 48 hours. Samples treated with ctx184+cer 131 showed significantly fewer tumor cells in culture after about 2 days when compared to samples treated with ctx184 alone or ctx184+ctx156 control T cells. All data were collected via FACS. Bottom of fig. 5B: representative flow charts of counts of JeKo-1 cells remaining after 48 hours co-culture. Top of fig. 5B: raw flow data from the bottom of FIG. 5B was used to calculate the bar graph of the remaining JeKo-1 cells. Fig. 5C: jeKo-1 cells were co-cultured with pCTX184 (1928 z) + pCTX131, pCTX184+CTX156 control T cells or pCTX184 cells alone at a ratio of 0.5:1T cells to JeKo-1 for 48 hours. Samples treated with ctx184+cer 131 showed increased IFN- γ secretion compared to samples treated with ctx184 alone or ctx184+ctx156 control T cells.

Fig. 6A-6B: pCTX131 chimeric Tim4 receptor-T cells enhance the potency of CD1928z CAR-T cells. Cytotoxic response evaluation CD1928z CAR-T (pCTX 184, also referred to as CAR 184) + pCTX131 chimeric Tim4 receptor-T cell mixture. Fig. 6A: pCTX131 chimeric Tim4 receptor T cells were combined with CD1928z CAR-T cells (pCTX 184) at different ratios and the JekO-1 cell counts were quantified over time. Fig. 6B: cysteine protease 3/7 response. All data were collected via incucyte.

Fig. 7A-7B: pCTX133 (a chimeric Tim4 receptor containing TLR-2) enhances the efficacy of nilaparib (Niraparib) in ovarian cancer models. Fig. 7A: flow cytometry measurement of surface PtdSer. Kuramochi cells were treated with 1.56 or 25 μm nilaparib or with an equivalent volume of DMSO (control). After 48 hours, the samples were trypsinized and stained with Tim4-Fc followed by a fluorescent-labeled secondary antibody against Tim 4-Fc. Fig. 7B: kuramochi cells pretreated with 1.56 μm nilaparib for about 20 hours were co-cultured with pCTX and untransduced CD 4T cells from donor 32 at a 2:1 ratio of T cells to Kuramochi and a final nilaparib concentration of 1.56 μm. Samples treated with nilaparib + pCTX exhibited substantially fewer tumor cells in culture after about 3 days when compared to samples treated with nilaparib alone or samples treated with nilaparib + non-transduced T cells. All data were collected via IncuCyte.

Fig. 8A-8B: chimeric Tim4 receptor-T cell + BTK inhibitor (ibrutinib) combination for hematological malignancies. Fig. 8A: ibrutinib induces the expression of phosphatidylserine on target cells. Fig. 8B: chimeric Tim4 receptor-T cell mediated synergistic cell killing in combination with BTK inhibitor small molecules. CTX136 (Tim 4-CD28-CD3 z) T cells co-cultured with e:t at 3:1, 2:1 and 1:1 in the presence of ibrutinib showed a significant increase in killing compared to empty vector transduced cells or ibrutinib treatment alone.

Fig. 9A-9B show transfection of Jurkat cells with various chimeric Tim4 receptor constructs pCTX1183, pCTX1161, pCTX1189, pCTX1184, pCTX1163, pCTX1162, pCTX1190, pCTX1186, pCTX1187, pCTX1164, pCTX1185, and pCTX 1165. Fig. 9B was normalized to untransfected cells.

FIG. 10 is a bar graph showing that activation of HPV E7 TCR T cells by antigen presentation mediated by chimeric Tim4-T cells is blocked by anti-HLA-I antibodies.

Fig. 11 depicts various chimeric Tim4 receptors (also referred to herein as chimeric phagocytic receptors (CER)) designed. SP = signal peptide; TMD = transmembrane domain.

FIGS. 12A-12B depict induction of phosphatidylserine exposure in REC-1 and JEKO-1 cells when treated with ibrutinib for 24 hours (FIG. 12A) and 48 hours (FIG. 12B).

FIGS. 13A-13D depict phagocytic activity of CER T cells on REC-1 cells after ibrutinib treatment. Fig. 13A: representative flow cytometry histograms of CER1234, CER1161 and CER 1183T cells phagocytosis of pHrodo red-labeled REC-1 target cells are shown. Fig. 13B: a graphical summary of flow cytometry data showing CER1234, CER1161 and CER 1183T cells phagocytosis of the ph rodo red-labeled REC-1 target cells. * P <0.01, < P <0.001, < P <0.0001; average + SD, n=4. Fig. 13C: representative fluorescence microscopy images (40-fold) of CER1234, CER1161 and CER 1183T cells phagocytose the ph rodo red-labeled REC-1 target cells are shown. Fig. 13D: phagocytosed pHrodo green-labeled REC-1 tumor fragments were co-localized to Lyso-tracker red-labeled lysosomes.

Fig. 14A-14B: cytotoxic activity against REC-1 cells by CER1183 and CER 1234T cells of ibrutinib. Fig. 14A: the vehicle pretreatment was followed by +/-0.5. Mu.M ibrutinib. Fig. 14B: mu.M ibrutinib pretreatment was followed by +/-0.5. Mu.M ibrutinib.

Fig. 15A-15B: cell surface markers that induce T cell activation in ibrutinib-treated co-cultures of CER1183 and CER 1234T cells with REC-1 target cells. The samples shown from left to right in the bar graph are untransduced CER-1183 and CER-1234. Fig. 15A: PD-1 expression in co-culture. Fig. 15B: 4-1BB expression in co-culture.

Fig. 16A-16H: cytokines were induced in ibrutinib-treated co-cultures of CER1183 and CER 1234T cells with REC-1 target cells. The samples shown from left to right in the bar graph are untransduced CER-1183 and CER-1234. Fig. 16A: tnfα induction. Fig. 16B: IL-2 induction. Fig. 16C: ifnγ. Fig. 16D: IL-5 induction. Fig. 16E: IL-6 induction. Fig. 16F: IL-10 induction. Fig. 16G: IL-4 induction. Fig. 16H: and (3) granzyme B induction.

Fig. 17A-17B: expression of CER 1236T cells. 6 days after transduction, CER 1236T cells readily expressed Tim4 on transduced T cells.

Fig. 18: CER T cell characteristics. Upper graph: the memory T cell subsets from top to bottom were: temra, tem, tcm, initial

Fig. 19A-19C: in a cell-free in vitro system, CER T cells are activated and produce IFN- γ in response to the involvement of phosphatidylserine. Fig. 19A: IFN-gamma production in response to Phosphatidylserine (PS). IFN- γ production was induced in CER-1236 and CER-1234T cells stimulated with PS (FIG. 19A), but not with Phosphatidylethanolamine (PE) (FIG. 19B). PS EC50 values for CER-1234 and CER-1236 from donors 38 and 41 are as follows: CER-1234 at 3.52 μg/mL (SE.+ -. 0.13) and 1.88 μg/mL (SE.+ -. 0.48), respectively; CER-1236 was 4.74. Mu.g/mL (SE.+ -. 0.02) and 3.16. Mu.g/mL (SE.+ -. 0.09), respectively. Fig. 19C: CER 1236T cells were able to perform recursive stimulation in vitro. Recursive IFN-gamma induction of 3 consecutive stimulations was performed at the indicated time points 24 hours after rest after the first stimulation.

Fig. 20: FACS and imaging-based detection of pHrodo dye-labeled tumor cell phagocytosis and endocytosis.

Fig. 21: CER 1236T cells showed increased phagocytosis by Jeko1 TMEM30a ^-/- cells (left panel-fluorescence microscopy image; right panel phagocytosis index value).

Fig. 22: CER1234 or CER 1236T cells showed cytotoxic function against Jeko1TMEM30a ^-/- MCL cells as measured by the incucyte assay.

Fig. 23: CER 1236T cells cooperate with ibrutinib to enhance killing of REC-1MCL cells.

Fig. 24: ibrutinib-induced phosphatidylserine exposure enhanced CER 1234T cytokine response (IFN- γ -left panel; granzyme B-right panel; untransduced left sample, CER-1234 right sample).

Fig. 25A-25B: CER 1236T cells activate E7 TCR T cells via presentation of tumor cell-derived E7 antigen in an HLA-I dependent manner. Fig. 25A: CER-1236, but not non-transduced T cells or anti-CD 19CAR T cells, induced HLA-DR on E7 TCR cells after co-culture with E7 oncoprotein positive/PS positive SCC152 target cells. Fig. 25B: in a separate experiment, HLA-DR induction on E7 TCR cells was blocked by HLA-I blocking antibodies but not by control isotype antibodies after CER-1236 co-culture with E7 oncoprotein positive/PS positive SCC152 cells. The bar graph depicts the percentage of E7 TCR T cells expressing the activation marker HLA-DR. Error bars show SEM.

Fig. 26: CER 1236T cells mediated antitumor effects in MCL xenografts (Jeko 1 TMEM30a ^-/- cells) (upper left). The upper right panel shows in vivo amplification data. The lower panel shows cytotoxicity data.

FIGS. 27A-27B show schematic diagrams of chimeric Tim4 receptors CER-1234 and CER1236 of the present disclosure (FIG. 27A); and (fig. 27B) a schematic of an assay established for assessing the immunophenotype of chimeric Tim4 receptor co-cultured with PARP inhibitor treated ovarian cancer cells. CER-1234 and CER1236 have an extracellular Tim4 binding domain, a CD28 transmembrane domain, a CD28 signaling domain, a TLR2 signaling domain, and a CD3 zeta signaling domain, and show the orientation of the signaling domains in the chimeric Tim4 receptor construct. Also depicted is a control molecule CER-1183 having a Tim4 extracellular binding domain, a Tim4 transmembrane domain, and a Tim4 signaling domain.

FIG. 28 shows various T cell products, multiplicity of infection (MOI) and% T cells as CER+ produced by transduction of donor T cells with CER-1234, CER-1236 and control CER-1183.

FIGS. 29A-29C show% CCR7+ expressing cells in Tim4+ T cells (T cells transduced with CER-1234, CER-1236 or control CER-1183) when chimeric Tim4 receptor transduced T cells, olaparib or Nilaparib were co-cultured with target A2780 ovarian cancer cells for 120 hours. The left to right samples on the bar graph are: a2780+ T cells, a2780+ T cells +nilaparib, and a2780+ T cells +olaparirib. Fig. 29A shows CCR7 expression on tim4+ T cells from donor 41. Fig. 29B shows CCR7 expression on tim4+ T cells from donor 38. Fig. 3C shows CCR7 expression on tim4+ T cells from donor 45. For FIGS. 29A-29C, for each donor T cell sample, conditions A2780+ T cells were co-cultured; a2780+ T cells + nilaparib; a27080+t cells+olaparib are shown from left to right in the bar graph.

FIGS. 30A-30C show CD4/CD 8T cell ratios when chimeric Tim4 receptor transduced T cells, olaparib or Nilapatinib were co-cultured with target A2780 ovarian cancer cells for 120 hours. The left to right samples on the bar graph are: a2780+ T cells, a2780+ T cells +nilaparib, and a2780+ T cells +olaparirib. FIG. 30A shows the CD4/CD 8T cell ratio in T cells from donor 41. FIG. 30B shows the CD4/CD 8T cell ratio in T cells from donor 38. FIG. 30C shows the CD4/CD 8T cell ratio in T cells from donor 45. For FIGS. 4A-4C, for each donor T cell sample, conditions A2780+ T cells were co-cultured; a2780+ T cells + nilaparib; a27080+t cells+olaparib are shown from left to right in the bar graph.

Fig. 31A-31B: PARP inhibitors induce phosphatidylserine on a2780 ovarian cancer cells. A2780 cells treated with nilaparib or olaparib (both 0.25 to 6 μm) had viability at 96 hours (fig. 31A) and Phosphatidylserine (PS) exposure (fig. 31B).

Fig. 32: CER T cell characterization.

Fig. 33A-33C: in a cell-free in vitro system, CER T cells are activated and produce IFN- γ in response to the involvement of phosphatidylserine. Fig. 33A: IFN-gamma production in response to Phosphatidylserine (PS). IFN- γ production was induced in CER-1236 and CER-1234T cells stimulated with PS (FIG. 33A), but not with Phosphatidylethanolamine (PE) (FIG. 33B). PS EC50 values for CER-1234 and CER-1236 from donors 38 and 41 are as follows: CER-1234 at 3.52 μg/mL (SE.+ -. 0.13) and 1.88 μg/mL (SE.+ -. 0.48), respectively; CER-1236 was 4.74. Mu.g/mL (SE.+ -. 0.02) and 3.16. Mu.g/mL (SE.+ -. 0.09), respectively. Fig. 33C: CER 1236T cells were able to perform recursive stimulation in vitro. Recursive IFN-gamma induction of 3 consecutive stimulations was performed at the indicated time points 24 hours after rest after the first stimulation.

Fig. 34A-34C: the combination of CER T cells with PARP inhibitors is cytotoxic to a2780 cells. Fig. 34A: a reduction in proliferation of a2780 cells was observed after 120 hours of co-culture with CER and PARPi, as measured by incucyte of 2 donors. Fig. 34B: at 120 hours, the ratio of cells remaining in the treated samples was quantified using the incucyte data from fig. 34A, compared to the untreated a2780 samples. Fig. 34C: proliferation of CER T cells in a co-culture assay.

Fig. 35A-35C: CER T cells produce cytokines in response to co-culture with PARP inhibitor treated a2780 ovarian cancer cells. The samples were from left to right: no drug, nilaparib treatment and olaparib treatment. FIG. 35A-IFN-y. FIG. 35B-TNF- α. FIG. 35C-granzyme B.

Fig. 36A-36B: CER 1236T cells activate E7 TCR T cells via presentation of tumor cell-derived E7 antigen in an HLA-I dependent manner. Fig. 36A: CER-1236, but not non-transduced T cells or anti-CD 19CAR T cells, induced HLA-DR on E7 TCR cells after co-culture with E7 oncoprotein positive/PS positive SCC152 target cells. Fig. 36B: in a separate experiment, HLA-DR induction on E7 TCR cells was blocked by HLA-I blocking antibodies but not by control isotype antibodies after CER-1236 co-culture with E7 oncoprotein positive/PS positive SCC152 cells. The bar graph depicts the percentage of E7 TCR T cells expressing the activation marker HLA-DR. Error bars show SEM.

Fig. 37: ibrutinib-induced phosphatidylserine exposure enhances CER 1236T cell IFN- γ responses. No response was seen for the untransduced cells. JeKo-1 MCL mCherry + cells were pretreated with 20 μm ibrutinib or vehicle for 48 hours. Jeko-1 MCL cells were then co-cultured with CER 1236T cells in serum-free medium +200IU/ml IL-2 and 0.5. Mu.M ibrutinib or vehicle. After 120 hours, IFN- γ secretion was measured using ELLA automated ELISA.

Fig. 38: CER 1236T cells had enhanced and more complete cytotoxicity on ibrutinib-treated REC-1MCL cells, but no enhanced and more complete cytotoxicity on untreated REC-1MCL cells.

Fig. 39: a graph showing NSCLC H1975 cell growth for cells pretreated with 100nM of octenib or vehicle and then co-cultured with CER 1236T cells.

Fig. 40: shows a graph (left to right: granzyme B, TNF α, IL-6 and ifnγ) of cytokine production by CER 1236T cells co-cultured with NSCLC H1975 cells with 100nM of octenib or vehicle.

Fig. 41: bar graphs showing the expansion fold of total cd3+ T cells (untransduced + vehicle, untransduced + octenib, CER1236+ vehicle, CER1236+ octenib) determined by quantitative flow cytometry using quantitative beads over a period of time (48 hours, 96 hours, 120 hours, and 144 hours).

Fig. 42: flow cytometry profiles of cd3+ cells (y-axis) and H1975 cells (x-axis) were measured in co-culture experiments of H1975 cells pre-treated with vehicle or of octenib (4 m 88nm,19.53 nm) and non-transduced T cells (top row) or CER 1236T cells (bottom row).

FIG. 43 is a schematic drawing depicting an assay design for measuring CER1236 reactivation in response to stimulation by repeating phosphatidylserine stimulation of CER 1236T cells and measurement of IFNγ production.

Fig. 44 is a graph showing ifnγ production of plate-bound phosphatidylserine by CER 1234T cells (left) or CER 1236T cells (right) from three donors in response to titrating doses.

Fig. 45 is a graph showing ifnγ production in CER 1234T cells or CER 1236T cells by donors in response to increasing doses of plate-bound phosphatidylserine.

Fig. 46 is a graph showing ifnγ production in CER 1234T cells or CER 1236T cells by donors in response to increasing doses of plate-bound phosphatidylserine on day 14 after thawing.

Fig. 47 is a graph showing T cell expansion (left), ifnγ production (center), and cell viability (right) in CER 1236T cells in response to increasing doses of plate-bound phosphatidylserine.

Fig. 48 is a graph showing T cell expansion (left), ifnγ production (center), and cell viability (right) in CER 1234T cells in response to increasing doses of plate-bound phosphatidylserine.

FIG. 49 is a schematic drawing depicting the design of an exemplary chimeric Tim4 construct for use in antigen presentation experiments.

FIG. 50 shows a bar graph measuring the T cell activation markers CD25 (left) and HLA-DR (right) as measured by flow cytometry. CER1183, CER1161, CER1234 or CER 1236T cells were cultured alone or co-cultured with phosphatidylserine positive HPV E7 oncoprotein positive cells (TMEM 30A-/-SCC 152) for 48 hours, followed by positive selection of T cells and co-culture with CELL TRACE violet labeled HPV E7 TCR T cells for 4 days. Error bars show SEM.

FIG. 51 shows a bar graph measuring the T cell activation marker HLA-DR as measured by flow cytometry. CER1183, CER1161, CER1234 or CER 1236T cells were cultured alone or co-cultured with phosphatidylserine positive HPV E7 oncoprotein positive cells (TMEM 30A-/-SCC 152) for 48 hours, followed by positive selection of T cells and co-culture with CELL TRACE violet-labeled HPV E7 TCR T cells for 4 days (fig. 51, left panel) or 6 days (fig. 51, right panel). Error bars show SEM.

FIG. 52 shows bar graphs measuring the T cell activation markers CD25 (left panel) and HLA-DR (right panel) as measured by flow cytometry. CER1183, CER1161, CER1234 or CER 1236T cells were cultured alone or co-cultured with phosphatidylserine positive HPV E7 oncoprotein positive cells (TMEM 30A-/-SCC 152) for 48 hours, followed by positive selection of T cells and co-culture with CELL TRACE violet labeled HPV E7 TCR T cells for 4 days. Error bars show SEM.

FIG. 53 shows a bar graph measuring the T cell activation marker HLA-DR as measured by flow cytometry. CER1183, CER1161, CER1234 or CER 1236T cells were cultured alone or co-cultured with phosphatidylserine positive HPV E7 oncoprotein positive cells (TMEM 30A-/-SCC 152) for 48 hours, followed by positive selection of T cells and co-culture with CELL TRACE violet labeled HPV E7 TCR T cells for 6 days. Error bars show SEM.

FIG. 54 shows bar graphs measuring the T cell activation markers CD25 (left panel) and HLA-DR (right panel) as measured by flow cytometry. CER1183, CER1161, CER1234 or CER 1236T cells were cultured alone or co-cultured with phosphatidylserine positive HPV E7 oncoprotein positive cells (TMEM 30A-/-SCC 152) for 48 hours, followed by positive selection of T cells and co-culture with CELL TRACE violet labeled HPV E7 TCR T cells for 4 days. Error bars show SEM.

Detailed Description

In one aspect, the present disclosure provides chimeric T cell immunoglobulin mucin 4 (Tim 4) receptors, also known as chimeric phagocytic receptors (CER). The chimeric Tim4 receptors of the disclosure confer phagocytic and/or cytotoxic activity to a host cell (e.g., T cell) modified with the chimeric Tim4 receptor, wherein the cytotoxic activity is induced upon binding of the chimeric Tim4 receptor to its target antigen phosphatidylserine. In some embodiments, the chimeric Tim4 receptor confers phagocytosis, cytotoxicity, and enhanced antigen capture, antigen processing, and antigen presentation activity to a modified host cell (e.g., a T cell).

In some embodiments, the chimeric Tim4 receptors described herein comprise a single-chain chimeric protein comprising: (a) An extracellular domain comprising a binding domain, the binding domain comprising: (i) a Tim4 IgV domain and a Tim4 mucin domain; (b) An intracellular signaling domain, wherein the intracellular signaling domain comprises a signaling domain comprising an immune receptor tyrosine activation motif (ITAM); a costimulatory signaling domain; and TLR signaling domains.

In some embodiments, the extracellular domain of a chimeric Tim4 receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain.

In some embodiments, the chimeric Tim4 receptor comprises a single-chain chimeric protein comprising: (a) an extracellular domain comprising a Tim4 binding domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain; and (c) a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. In certain embodiments, the extracellular domain of a chimeric Tim4 receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain. In certain embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17.

In some embodiments, the chimeric Tim4 receptor comprises a single chain chimeric protein comprising, from N-terminus to C-terminus: (a) an extracellular domain comprising a Tim4 binding domain; (b) a CD28 transmembrane domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, (c) a CD3 zeta signaling domain, and (d) a TLR2 signaling domain. In certain embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17. In some embodiments, such chimeric Tim4 receptors comprise or consist of the amino acid sequence set forth in SEQ ID No. 19.

In some embodiments, the chimeric Tim4 receptor comprises a single chain chimeric protein comprising, from N-terminus to C-terminus: (a) an extracellular domain comprising a Tim4 binding domain; (b) a CD28 transmembrane domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, (c) a TLR2 signaling domain, and (d) a CD3 zeta signaling domain. In certain embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17. In some embodiments, such chimeric Tim4 receptors comprise or consist of the amino acid sequence set forth in SEQ ID No. 18.

In some embodiments, the chimeric Tim4 receptors of the present disclosure also confer phagocytic activity on a host cell when expressed in a host cell, such as a T cell. For example, in certain such embodiments, binding of the chimeric Tim4 receptor expressed in a host cell to a phosphatidylserine target can induce a cytolytic and phagocytic response by the host cell. In particular embodiments of the modified host cells described herein, the host cells do not naturally exhibit a phagocytic phenotype prior to modification with the chimeric Tim4 receptor.

In another aspect, host cells modified with the chimeric Tim4 receptors of the present disclosure can be used in methods of eliminating target cells with surface exposed phosphatidylserine, e.g., for treating cancer. In normal healthy cells, phosphatidylserine is located in the inner leaflet of the plasma membrane. However, certain cellular events (such as injury, apoptosis, necrosis, and stress) activate a "promiscuous enzyme (scramblase)", which rapidly exposes phosphatidylserine to the cell surface where it binds to receptors such as Tim 4. Endogenous tumor-specific effector T cells can induce exposure of phosphatidylserine on the outer membrane of targeted tumor cells during cell lysis. In addition, certain cancer therapies (e.g., chemotherapy, molecular targeted therapies, radiation therapies, CAR-T cells, etc.) can induce phosphatidylserine exposure to targeted tumor cells or cells in the tumor microenvironment by inducing apoptosis, cellular stress, cellular injury, etc. Engineered host cells expressing the disclosed chimeric Tim4 receptors can clear damaged, stressed, apoptotic or necrotic tumor cells bearing surface-exposed phosphatidylserine by inducing apoptosis in tumor cells bearing surface-exposed phosphatidylserine. In certain embodiments, host cells expressing the chimeric Tim4 receptors disclosed herein clear damaged, stressed, apoptotic, or necrotic tumor cells with surface-exposed phosphatidylserine by inducing apoptosis and by phagocytosis. An engineered host cell comprising a chimeric Tim4 receptor according to the present description may be administered to a subject alone or in combination with one or more additional therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapy, molecular targeted therapies, small molecules, oncolytic viruses, electric pulse therapies, and the like.

In another aspect, host cells modified with the chimeric Tim4 receptors of the present disclosure can be used in methods of enhancing effector responses (e.g., tumor-specific immune responses). In embodiments, host cells modified with the chimeric Tim4 receptors of the present disclosure can be used in methods for enhancing anti-tumor efficacy (e.g., tumor trafficking, expansion, and persistence). Embodiments of the chimeric Tim4 receptors of the present disclosure are capable of costimulating T cells via at least one costimulatory signaling pathway upon binding to phosphatidylserine. In certain embodiments, the chimeric Tim4 receptors described herein provide a co-stimulatory signal via at least two different signaling pathways. In certain embodiments, the enhanced effector response is enhanced T cell proliferation, cytokine production, cytotoxic activity, persistence, or any combination thereof. Host cells expressing a chimeric Tim4 receptor according to the present description can be administered to a subject alone or in combination with one or more additional therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapy, small molecules, oncolytic viruses, electric pulse therapy, and the like.

In another aspect, host cells modified with the chimeric Tim4 receptors of the present disclosure can be used in methods of inhibiting or reducing immune cell depletion. In certain embodiments, immune cell depletion refers to T cell depletion, NK cell depletion, or both. Tumor cells can generally provide sustained antigen stimulation to immune cells in the absence of co-stimulatory ligands, which can lead to immune cell depletion (e.g., reduced proliferation capacity, reduced effector function, and up-regulation of immunosuppressive molecules). Cancer treatment (e.g., chemotherapy, molecular targeted therapy, radiation therapy, CAR-T cell therapy, etc.) may also provide prolonged antigen stimulation in the absence of a co-stimulatory signal or when the intensity or duration of the co-stimulatory signal is limited. The chimeric Tim4 receptors of the present disclosure are capable of costimulating immune cells via at least one costimulatory signaling domain when bound to phosphatidylserine. In certain embodiments, the chimeric Tim4 receptor provides a co-stimulatory signal via at least two different signaling pathways. Host cells expressing the chimeric Tim4 receptor can be administered to a subject alone or in combination with one or more additional therapeutic agents, including, for example, CAR-T cells, TCRs, antibodies, radiation therapy, chemotherapy, small molecules, oncolytic viruses, electric pulse therapy, and the like.

In some embodiments, host cells (e.g., T cells) modified with the chimeric Tim4 receptors of the present disclosure exhibit enhanced antigen capture, antigen processing, and/or antigen presentation activity. Ligand binding of the phagocytic receptor portion of the chimeric Tim4 receptor mediates a series of events, including: t cell activation, signal transduction, cytolytic function, production of cytokines and chemokines, partial phagocytosis of target cells and downstream transcription processes leading to presentation of target cell antigens. Expression of the chimeric Tim4 receptor in non-phagocytic or poorly phagocytic immune cells (e.g., mature polyclonal T cells) can be achieved by phagocytizing the target cell fragment and enhancing antigen-specific capture. In some embodiments, the increased functionality of chimeric Tim4 receptor-mediated antigen capture supports enhanced presentation of non-targeted antigens while also eliciting direct cytolytic activity against sensitized tumor cell targets.

For combination therapy compositions and methods comprising a chimeric Tim4 receptor and a cellular immunotherapy (e.g., CAR or TCR) according to the present description, the chimeric Tim4 receptor and the cellular immunotherapeutic (e.g., CAR or TCR) can be expressed on separate engineered cells or on the same engineered cells to produce dual-specific, multifunctional engineered cells. The chimeric Tim4 receptor and the cellular immunotherapeutic expressed on the same engineered cell may be expressed from separate vectors, or expressed on the same vector in the form of a polycistronic construct.

In another aspect, host cells modified with the chimeric Tim4 receptors of the present disclosure can be used to enhance the effect of therapeutic agents that induce cellular stress, injury, necrosis, or apoptosis. For example, certain therapeutic agents, such as chemotherapy, specific inhibitors of driver gene mutations associated with cancer (molecular targeted therapies such as BRAF inhibitors, EGFR inhibitors, ALK/ROS1 kinase inhibitors, BTK inhibitors, PARP inhibitors), radiation therapy, UV light therapy, electrical pulse therapy, adoptive cellular immunotherapy (e.g., CAR-T cells, TCRs) and oncolytic virus therapy, can induce cell damage or death in tumor cells or diseased cells. Cells expressing the chimeric Tim4 receptor as described herein can bind to phosphatidylserine moieties on the outer leaflet of damaged or dead cells produced by any one or more of such therapeutic agents and induce cytolysis or both cytolysis and phagocytosis of the targeted cells.

In another aspect, the present disclosure provides a method for enhancing ccr7+ expressing T cells in a subject having cancer, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer or melanoma, comprising administering to the subject an effective amount of a chimeric Tim4 receptor according to the present description and (ii) a poly ADP-ribose polymerase (PARP) inhibitor. In some embodiments, the chimeric Tim4 receptor comprises: (a) An extracellular domain comprising a Tim4 binding domain, (b) an intracellular signaling domain comprising a CD28 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain; and (c) a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. In certain embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17. Chemokine receptor CCR7 is expressed on T cells in early differentiation states, such as naive T cells, stem cell memory (T _SCM) T cells, and central memory (T _CM) T cells. Early preclinical studies on therapeutic T cells showed that both naive T cells and early differentiated T cells have enhanced long-term persistence and can elicit potent anti-tumor responses. Thus, enhancement of CCR7 chimeric Tim4 receptor T cells may also enhance implantation and persistence in vivo.

In another aspect, the present disclosure provides a method for increasing the CD4/CD 8T cell ratio in a subject having cancer, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer or melanoma, comprising administering to the subject an effective amount of a chimeric Tim4 receptor according to the present description and optionally a poly ADP-ribose polymerase (PARP) inhibitor. In some embodiments, the chimeric Tim4 receptor comprises: (a) an extracellular domain comprising a Tim4 binding domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain; and (c) a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. In certain embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17. Helper functions of CD 4T cells are known, and cytolytic activity is induced by enhancing the activity of CD 8T cells via cytokine production. However, adoptive cell therapy using CD 4T cells (chimeric antigen receptor (CAR)) has shown considerable effectiveness in directly killing target tumor cells both in vitro and in vivo. Whereas adoptive cell therapy with CD 4T Cells (CARs) showed initially slower granzyme B secretion and tumor killing, but less susceptible to activation-induced cell death and failure compared to CD8 counterparts, which resulted in CD4 CAR T cells with relatively better persistence after antigen exposure. Thus, enhancing a CD 4T cell subpopulation of chimeric Tim4 receptor adoptive cell therapies may confer improved persistence and less failure.

In another aspect, host cells expressing the chimeric Tim receptors disclosed herein clear damaged tumor cells, stressed tumor cells, apoptotic tumor cells, or necrotic tumor cells with surface exposed phosphatidylserine by inducing apoptosis and by phagocytosis. In another aspect, host cells modified with the chimeric Tim receptors of the present disclosure can be used to enhance the effect of PARP inhibitors that induce cellular stress, injury, necrosis, or apoptosis. For example, administration of PARP inhibitors can increase the level of surface phosphatidylserine, resulting in a synergistic combination. The presently described cells expressing the chimeric Tim receptor can bind to the exposed phosphatidylserine moiety on the outer leaflet of damaged or dead cells resulting from the administration of PARP inhibitors and induce cytolysis or both cytolysis and phagocytosis of the targeted cells.

In another aspect, the present disclosure provides a method for treating cancer in a subject comprising administering a chimeric Tim4 receptor in combination with a poly (ADP-ribose) polymerase (PARP) inhibitor. The chimeric Tim4 receptor can be administered as a composition comprising a chimeric Tim receptor, a polynucleotide encoding a chimeric Tim4 receptor, a vector comprising a polynucleotide encoding a chimeric Tim4 receptor, or an engineered host cell comprising a chimeric Tim4 receptor, a polynucleotide encoding a chimeric Tim4 receptor of a vector comprising a polynucleotide encoding a chimeric Tim4 receptor, wherein optionally the composition further comprises a pharmaceutically acceptable excipient. PARP inhibitors may be administered in sub-therapeutic doses. In another aspect, the present disclosure provides a pharmaceutical composition or combination comprising a chimeric Tim4 receptor and a PARP inhibitor. The chimeric Tim4 receptors useful in the compositions and methods of the present disclosure confer phagocytic, cytotoxic, and/or antigen presenting activity on a chimeric Tim4 receptor-modified host cell (e.g., a T cell), wherein the cytotoxic activity is induced upon binding of the chimeric Tim4 receptor to its target antigen phosphatidylserine. DNA damaging agents such as PARP inhibitors can induce effector functions of chimeric Tim4 receptors, such as phagocytosis, cytotoxicity, co-stimulatory activity, antigen presentation, or combinations thereof, by promoting cellular damage and the extravasation of phosphatidylserine to act synergistically with the chimeric Tim4 receptor.

Before setting forth the present disclosure in more detail, it may be helpful to understand the present disclosure to provide definitions of certain terms to be used herein.

In this description, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range and to include fractions thereof (e.g., tenths and hundredths of integers) as appropriate. Furthermore, any numerical range recited herein in connection with any physical feature, such as a polymer subunit, size, or thickness, should be understood to include any integer within the range, unless otherwise indicated. As used herein, unless otherwise indicated, the term "about" means ± 20% of the indicated range, value, or structure. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the recited components. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof. As used herein, the terms "comprising," "having," and "including" are used synonymously, and these terms and variants thereof are intended to be construed as non-limiting.

Unless defined otherwise herein, terms are to be understood by those skilled in the art of antibodies in the sense given in the art. The term "antibody" is used in the broadest sense and encompasses polyclonal antibodies and monoclonal antibodies. An "antibody" may refer to an intact antibody that includes at least two heavy (H) and two light (L) chains linked to each other by disulfide bonds, as well as an antigen-binding portion (or antigen-binding domain) of the intact antibody that has or retains the ability to bind to a target molecule. Antibodies can be naturally occurring, recombinantly produced, genetically engineered, or modified forms of immunoglobulins, e.g., intracellular antibodies, peptibodies, nanobodies, single domain antibodies, SMIPs, multispecific antibodies (e.g., bispecific antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, tandem bis-scFV, tandem tri-scFV, ADAPTIR). The monoclonal antibody or antigen binding portion thereof may be non-human, chimeric, humanized or human, preferably humanized or human. Antibodies are described, for example, in Harlow et al: the structure and function of immunoglobulins is reviewed in the laboratory Manual (Antibodies: A Laboratory Manual), chapter 14, (Cold spring harbor laboratory (Cold Spring Harbor Laboratory), cold spring harbor (Cold Spring Harbor), 1988). The "antigen binding portion" or "antigen binding domain" of an intact antibody is intended to encompass such "antibody fragments": which represents a portion of an intact antibody and refers to the epitope variable region or complementarity determining region of the intact antibody. Examples of antibody fragments include, but are not limited to, fab ', F (ab') ₂, and Fv fragments, fab '-SH, F (ab') ₂, diabodies, linear antibodies, scFv antibodies, VH, and multispecific antibodies formed from antibody fragments. "Fab" (antigen binding fragment) is a portion of an antibody that binds to an antigen and comprises the variable region of the heavy chain linked to the light chain via an interchain disulfide bond and CH1. Antibodies can be of any class or subclass, including IgG and its subclasses (IgG ₁、IgG₂、IgG₃、IgG₄), igM, igE, igA, and IgD.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three CDRs. (see, e.g., kit et al, kuby Immunology, 6 th edition, w.h. frieman company (w.h. freeman and co.) (page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated from antibodies that bind that antigen using VH or VL domains to screen libraries of complementary VL or VH domains, respectively. See, e.g., portolano et al, J.Immunol.), 150:880-887 (1993); clarkson et al, nature, 352:624-628 (1991).

The terms "complementarity determining region" and "CDR," synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous amino acid sequences within the variable region of an antibody that confer antigen specificity and/or binding affinity. In general, there are three CDRs (HCDR 1, HCDR2, HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, LCDR 3) in each light chain variable region.

As used herein, the terms "binding domain," "binding region," and "binding moiety" refer to a molecule (such as a peptide, oligopeptide, polypeptide, or protein) that has the ability to specifically and non-covalently bind, associate (unite), recognize, or combine a target molecule (e.g., phosphatidylserine). The binding domain comprises any naturally occurring, synthetic, semisynthetic or recombinantly produced binding partner for a biomolecule or other target of interest. In some embodiments, the binding domain is an antigen binding domain, such as an antibody or a functional binding domain or antigen binding portion thereof. Exemplary binding domains include single chain antibody variable regions (e.g., domain antibodies, sFv, scFv, fab), receptor extracellular domains (e.g., tim 4), ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their ability to specifically bind to a biological molecule.

"T cell receptor" (TCR) refers to a molecule that is present on the surface of T cells (also known as T lymphocytes) and is generally responsible for recognizing antigens that bind to Major Histocompatibility Complex (MHC) molecules. In most T cells, TCRs are typically composed of heterodimers of disulfide-linked highly variable alpha and beta chains (also referred to as tcra and tcrp, respectively). In a small fraction of T cells, TCRs consist of heterodimers of gamma and delta chains (also referred to as tcrgamma and tcrdelta, respectively). Each chain of TCR is a member of The immunoglobulin superfamily and has an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region and a short cytoplasmic tail at The C-terminus (see Janeway et al, immunobiology: immune system in health and disease (Immunobiology: the Immune SYSTEMIN HEALTH AND DISEASE), 3 rd edition, contemporary biological publication (Current Biology Publications), page 4: 33, 1997). The TCRs of the present disclosure may be from a variety of animal species, including humans, mice, rats, cats, dogs, goats, horses, or other mammals. TCRs may be cell-bound (i.e., have a transmembrane region or domain) or soluble in form. TCRs include recombinantly produced, genetically engineered, fused or modified forms of TCRs, including, for example, sctcrs, soluble TCRs, TCR fusion constructs (TRuC ^TM; see, U.S. patent publication No. 2017/0166622).

The terms vy and vδ of the "variable region" or "variable domain" or γδ TCR of the TCR α chain (vα) and β chain (vβ) are involved in the binding of the TCR to the antigen. V _α and V _β of native TCRs generally have similar structures, where each variable domain includes four conserved FRs and three CDRs. The V _α domain is encoded by two separate DNA segments: a variable gene segment (V gene) and a junction gene segment (J gene); the V _β domain is encoded by three separate DNA segments: variable gene segments (V genes), diverse gene segments (D genes), and junction gene segments (J genes). A single V _α or V _β domain may be sufficient to confer antigen binding specificity. "major histocompatibility complex molecule" (MHC molecule) refers to a glycoprotein that delivers peptide antigens to the cell surface. MHC class I molecules are heterodimers composed of a membrane spanning the alpha chain (containing three alpha domains) and non-covalently associated beta 2 microglobulin. MHC class II molecules consist of two transmembrane glycoproteins, α and β, which both transmembrane. Each chain has two domains. MHC class I molecules deliver cytosolic derived peptides to the cell surface where the peptides: the MHC complex is recognized by CD8 ⁺ T cells. MHC class II molecules deliver peptides derived from the vesicle system to the cell surface where they are recognized by cd4+ T cells. MHC molecules may be from a variety of animal species, including humans, mice, rats or other mammals.

"Chimeric antigen receptor" (CAR) refers to a chimeric protein comprising two or more distinct domains and can act as a receptor when expressed on the surface of a cell. CARs generally consist of an extracellular domain (comprising a binding domain that binds a target antigen), an optional extracellular spacer domain, a transmembrane domain, and an intracellular signaling domain (e.g., a T cell activation motif containing an immune receptor tyrosine activation motif (ITAM), and optionally an intracellular co-stimulatory domain). In certain embodiments, the intracellular signaling domain of the CAR has a T cell activation domain (e.g., cd3ζ) and an intracellular co-stimulatory domain (e.g., CD 28) that contains ITAM. In certain embodiments, the CAR is synthesized as a single polypeptide chain, or encoded by a nucleic acid molecule as a single-chain polypeptide.

Various assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, and determining affinity of the binding domains, such as western blotting, ELISA, andAnalysis (see, e.g., scatchard et al, new York academy of sciences annual book (Ann. N.Y. Acad. Sci.) 51:660, 1949, and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalent). As used herein, "specifically binds" refers to a binding domain or fusion protein thereof that associates or associates with a target molecule with an affinity equal to or greater than 10 ⁵M^-1 or K _a (i.e., the equilibrium association constant of a particular binding interaction, where the units are 1/M), but does not significantly associate or associate with any other molecule or component in the sample.

The terms "antigen" and "Ag" refer to molecules capable of inducing an immune response. The immune response induced may be involved in antibody production, activation of specific immunocompetent cells, or both. Macromolecules (including proteins, glycoproteins and glycolipids) can be used as antigens. The antigen may be derived from recombinant DNA or genomic DNA. As contemplated herein, an antigen need not be encoded (i) by only the full length nucleotide sequence of the gene or (ii) by the "gene" at all. The antigen may be produced or synthesized, or the antigen may be from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

The term "epitope" or "antigenic epitope" encompasses any molecule, structure, amino acid sequence, or protein determinant within an antigen that is specifically bound by a cognate immune binding molecule, such as an antibody or fragment thereof (e.g., scFv), T Cell Receptor (TCR), chimeric Tim4 receptor, or other binding molecule, domain, or protein. Epitope determinants generally contain chemically active surface groupings of molecules such as amino acids or sugar side chains and may have specific three dimensional structural characteristics as well as specific charge characteristics. The epitope may be a linear epitope or a conformational epitope.

As used herein, the term "Tim4" (T cell immunoglobulin and mucin domain containing protein 4), also referred to as "TimD4", refers to the phosphatidylserine receptor that is normally expressed on antigen presenting cells such as macrophages and dendritic cells. Tim4 mediates phagocytosis by apoptotic, necrotic, injured or stressed cells that present phosphatidylserine (PtdSer) on the outer (external) leaflet of the cell membrane. Tim4 is also capable of binding to Tim1 expressed on the surface of T cells and inducing proliferation and survival. In certain embodiments, tim4 refers to human Tim4. An exemplary human Tim4 protein includes the amino acid sequence of SEQ ID NO. 1.

As used herein, the term "Tim4 binding domain" refers to the N-terminal immunoglobulin folding domain of Tim4, which has a metal ion-dependent pocket that selectively binds PtdSer. An exemplary human Tim4 binding domain comprises the amino acid sequence of SEQ ID NO. 5, and an exemplary mouse Tim4 binding domain comprises the amino acid sequence of SEQ ID NO. 2.

The Tim4 binding domain comprises a variable immunoglobulin (IgV) -like domain (referred to herein as an "IgV domain") and a mucin-like domain (referred to herein as an "mucin domain"). An exemplary human Tim4 IgV domain comprises the amino acid sequence of SEQ ID NO. 3, and an exemplary human Tim4 mucin domain comprises the amino acid sequence of SEQ ID NO. 4. In certain embodiments, the Tim4 binding domain does not comprise a signal peptide. An exemplary human Tim4 signal peptide has the amino acid sequence of SEQ ID NO. 7. An exemplary mouse Tim4 signal peptide has the amino acid sequence of SEQ ID NO. 8.

As used herein, an "effector domain" is an intracellular portion of a fusion protein or receptor that, upon receipt of an appropriate signal, can directly or indirectly promote a biological or physiological response in a cell expressing the effector domain. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule that triggers a signal from the effector domain. When the effector domain contains one or more signaling domains or motifs, such as an immune receptor tyrosine activation motif (ITAM), it may directly promote a cellular response. In other embodiments, the effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote the cellular response.

As used herein, a "costimulatory signaling domain" refers to an intracellular signaling domain of a costimulatory molecule, or a functional portion thereof, that, when activated together with a primary or classical (e.g., ITAM-driven) activation signal (e.g., provided by a cd3ζ intracellular signaling domain), promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. The costimulatory signaling domain comprises, for example ,CD27、CD28、CD40L、GITR、NKG2C、CARD1、CD2、CD7、CD27、CD30、CD40、CD54(ICAM)、CD83、CD134(OX-40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD226、CD270(HVEM)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、DAP10、LAT、LFA-1、LIGHT、NKG2C、SLP76、TRIM, or any combination thereof.

As used herein, an "immunoreceptor tyrosine-activating motif (ITAM) activation domain" refers to an intracellular signaling domain or functional portion thereof that is naturally or endogenously present on an immune cell receptor or cell surface marker and contains at least one immunoreceptor tyrosine-activating motif (ITAM). ITAM refers to the conserved motif YXXL/I-X _6-8 -YXXL/I. In certain embodiments, the ITAM signaling domain contains one, two, three, four, or more ITAMs. The ITAM signaling domain may initiate T cell activation signaling following antigen binding or ligand binding. The ITAM signaling domain comprises intracellular signaling domains such as cd3γ, cd3δ, cd3ε, cd3ζ, CD79a, and CD66 d.

"Connecting amino acid" or "connecting amino acid residues" refers to the polypeptide of two adjacent motifs, region or domain between one or more (e.g., about 2-20) amino acid residues. The linking amino acids may be from the construct design of the chimeric protein (e.g., amino acid residues generated using restriction enzyme sites during construction of the nucleic acid molecule encoding the chimeric protein).

"Nucleic acid molecules" and "polynucleotides" may be in the form of RNA or DNA, including cDNA, genomic DNA, and synthetic DNA. The nucleic acid molecule may consist of naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., the alpha-enantiomeric form of a naturally-occurring nucleotide), or a combination of both. The modified nucleotide may have a modification or substitution of a sugar moiety or a pyrimidine or purine base moiety. Nucleic acid monomers may be linked by phosphodiester bonds or analogues of such bonds. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, phosphoroselenites, phosphorodiselenates, phosphoroanilide, phosphorophosphoramidates, and the like. The nucleic acid molecule may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding strand (antisense strand). The coding molecules may have the same coding sequence as known in the art, or may have a different coding sequence which may encode the same polypeptide due to redundancy or degeneracy of the genetic code, or by splicing.

"Coding" refers to the inherent properties of specific polynucleotide sequences (e.g., DNA, cDNA, and mRNA sequences) to serve as templates for the synthesis of other polymers and macromolecules having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the biological properties that result therefrom during bioprocessing. Thus, if transcription and translation of mRNA corresponding to a polynucleotide produces a protein in a cell or other biological system, the polynucleotide encodes the protein. Both coding and non-coding strands may be referred to as encoding proteins or other products of polynucleotides. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" encompasses all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. The protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can make up the protein sequence or peptide sequence. A polypeptide comprises any peptide or protein consisting of two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as proteins, of many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptide comprises a natural peptide, a recombinant peptide, a synthetic peptide, or a combination thereof.

As used herein, the term "mature polypeptide" or "mature protein" refers to a protein or polypeptide secreted or localized in the cell membrane or within certain cellular organelles (e.g., endoplasmic reticulum, golgi, or endosomes), and does not comprise an N-terminal signal peptide.

"Signal peptide", also known as "signal sequence", "leader peptide", "localization signal" or "localization sequence", is a short peptide (typically 15-30 amino acids in length) that is present at the N-terminus of a newly synthesized protein and is transported to the secretory pathway. The signal peptide typically comprises a short, hydrophilic, positively charged amino acid at the N-terminus, a central hydrophobic domain of 5-15 residues and a C-terminal region with a cleavage site for the signal peptidase. In eukaryotes, the signal peptide facilitates the transport of the newly synthesized protein to the endoplasmic reticulum where it is cleaved by the signal peptidase to produce the mature protein, which then enters its appropriate destination.

The term "chimeric" refers to any nucleic acid molecule or protein that is non-endogenous and includes sequences that bind or join together (typically do not bind or join together in nature). For example, a chimeric nucleic acid molecule may include regulatory sequences and coding sequences from different sources, or regulatory sequences and coding sequences from the same source but arranged in a manner different from that found in nature.

As used herein, the term "endogenous" or "native" refers to a gene, protein, compound, molecule, or activity that is normally present in a host or host cell, including naturally occurring variants of the gene, protein, compound, molecule, or activity.

As used herein, "homologous" or "homolog" refers to a molecule or activity from a host cell that is related to a second gene or activity, e.g., from the same host cell, a different organism, a different strain, a different species. For example, a heterologous molecule or a heterologous gene encoding the molecule may be homologous to the native host cell molecule or the gene encoding the molecule, respectively, and may optionally have an altered structure, sequence, expression level, or any combination thereof.

As used herein, a "heterologous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to the host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of the heterologous nucleic acid molecule, construct or sequence may be from a different genus or species. In some embodiments, the heterologous nucleic acid molecule is not naturally occurring. In certain embodiments, the heterologous nucleic acid molecule is added (i.e., non-endogenous or native) to the host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, etc., wherein the added molecule may be integrated into the host cell genome or present as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies. Furthermore, "heterologous" refers to a non-native enzyme, protein, or other activity encoded by a non-endogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity.

As used herein, the term "engineered", "recombinant", "modified" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule or vector that has been modified by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism that has been genetically engineered by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, wherein such alterations or modifications may be introduced by genetic engineering. A human-produced genetic alteration may comprise, for example, the introduction of a modification of a nucleic acid molecule encoding one or more proteins, chimeric receptors or enzymes (which may comprise an expression control element, such as a promoter), or the addition, deletion, substitution of other nucleic acid molecules, or other functional disruption or addition of genetic material of a cell. Exemplary modifications include those in the coding region of a heterologous or homologous polypeptide from a reference or parent molecule, or a functional fragment thereof. Other exemplary modifications include, for example, modifications in non-coding regulatory regions, wherein the modifications alter expression of the gene or operon.

As used herein, the term "transgene" refers to a gene or polynucleotide encoding a protein of interest (e.g., chimeric Tim4 receptor), whose expression in a host cell is desired, and which has been transferred into the cell by genetic engineering techniques. The transgene may encode a protein of therapeutic interest, as a reporter, tag, marker, suicide protein, or the like. The transgene may be from a natural source, a modified or recombinant molecule of a natural gene, or a synthetic molecule. In certain embodiments, the transgene is a component of a vector.

The term "overexpression" or "overexpression" of an antigen refers to an abnormally high level of antigen expression in a cell. The overexpression of an antigen or antigen is often associated with a disease state, such as in hematological malignancies and cells that form solid tumors within a particular tissue or organ of a subject. Solid tumors or hematological malignancies characterized by overexpression of tumor antigens can be determined by standard assays known in the art.

The "percent identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity =number of identical positions/total number of positions×100), taking into account the number of gaps and the length of each gap that needs to be introduced to optimize alignment of the two or more sequences. Comparison of sequences and determination of percent identity between two or more sequences may be accomplished using mathematical algorithms, such as BLAST and Gapped BLAST programs using default parameters (e.g., altschul et al, (J. Mol. Biol.), 215:403, 1990; see also BLASTN of www.ncbi.nlm.nih.gov/BLAST).

"Conservative substitutions" are considered in the art to be amino acid substitutions of one amino acid for another having similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433, page 10, published 3/13 1997; lehninger, biochemistry, second edition; walsh Publishers, inc.) New York (1975), pages 71-77; lewis, genes IV, oxford university Press (Oxford University Press), new York and cell Press (CELL PRESS), cambridge, massachusetts (1990), page 8).

As used herein, the term "promoter" is defined as a DNA sequence recognized by a cellular or introduced synthetic mechanism that is required to initiate specific transcription of a polynucleotide sequence.

As used herein, the term "promoter/regulatory sequence" means a nucleic acid sequence required for expression of a gene product operably linked to a promoter/regulatory sequence. In some cases, the sequence may be a core promoter sequence, and in other cases, the sequence may also comprise enhancer sequences and other regulatory elements required for expression of the gene product. For example, the promoter/regulatory sequence may be one that expresses the gene product in a tissue specific manner.

A "constitutive" promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product under most or all physiological conditions of a cell.

An "inducible" promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or specifying a gene product, results in the production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded by or specified by a gene, results in the production of a gene product in a cell, essentially only if the cell is a cell of the tissue type corresponding to the promoter.

As used herein, the phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct position and orientation relative to the polynucleotide to control transcription by RNA polymerase and initiation of expression of the polynucleotide.

A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. The vector may be, for example, a plasmid, cosmid, virus or phage. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells. An "expression vector" refers to a vector that, when present in an appropriate environment, is capable of directing the expression of a protein encoded by one or more genes carried by the vector.

In certain embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, gamma retrovirus vectors, and lentiviral vectors. A "retrovirus" is a virus having an RNA genome. "Gamma retrovirus" refers to a genus of the family retrovirus. Examples of gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "lentivirus" refers to a genus of retrovirus capable of infecting dividing cells and non-dividing cells. Examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2), equine infectious anemia virus (FIV), bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

In other embodiments, the vector is a non-viral vector. Examples of non-viral vectors include lipid-based DNA vectors, modified mRNA (modRNA), self-amplified mRNA, closed-end linear duplex (CELiD) DNA, and transposon mediated gene transfer (PiggyBac). In the case of non-viral delivery systems, the delivery vehicle may be a liposome. The lipid formulation may be used to introduce nucleic acids into host cells in vitro, ex vivo, or in vivo. The nucleic acid may be encapsulated within the liposome, interspersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with the liposome and the nucleic acid, contained within or complexed with the micelle, or otherwise associated with the lipid.

As used herein, the term "phagocytosis" refers to receptor-mediated processes in which endogenous or exogenous cells or particles having a diameter greater than 100nm are internalized by the phagocytes or host cells of the present disclosure. Phagocytosis typically consists of multiple steps: (1) Binding the target cell or particle via direct or indirect (via bridging molecules) binding of the phagocytic receptor to a pro-phagocytic or antigenic marker on the target cell or particle; and (2) internalization or phagocytosis of the entire target cell or particle or a portion thereof. In certain embodiments, internalization may occur via cytoskeletal rearrangement of the phagocyte or host cell to form phagosome, i.e., a membrane-bound compartment containing the internalized target. Phagocytosis may further comprise maturation of phagosome, wherein phagosome becomes more and more acidic and fuses with lysosomes (to form phagolysosomes), whereby the target being phagocytosed is degraded (e.g., "phagocytosis"). Alternatively, phagosome-lysosomal fusion may not be observed during phagocytosis. In yet other embodiments, the phagosome may reflux or drain its contents into the extracellular environment before complete degradation. In some embodiments, phagocytosis refers to phagocytosis. In some embodiments, phagocytes include the binding of target cells or particles by phagocytes of host cells of the present disclosure, but do not include internalization. In some embodiments, phagocytosis comprises the binding of a target cell or particle by a phagocyte of a host cell of the present disclosure and internalization of a portion of the target cell or particle.

As used herein, the term "phagocytosis" refers to the process of phagocytosis of cells or large particles (> 0.5 μm) in which binding of target cells or particles, phagocytosis of target cells or particles, and degradation of internalized target cells or particles occurs. In certain embodiments, phagocytosis comprises the formation of phagosome that encompasses internalized target cells or particles, and the phagosome fuses with lysosomes to form phagosome, wherein the contents of the phagosome are degraded. In certain embodiments, during phagocytosis, a chimeric Tim4 receptor expressed on a host cell of the disclosure forms a phagocytic synapse upon binding to phosphatidylserine expressed by a target cell or particle; producing an actin-rich phagocytic cup at the phagocytic synapse; phagocytic arms extend around target cells or particles through cytoskeletal rearrangement; and eventually, the target cell or particle is pulled into the phagocytic cell or host cell by the force generated by the motor protein. As used herein, "phagocytosis" encompasses the process of "cytoburial", which specifically refers to phagocytosis of apoptotic or necrotic cells in a non-inflammatory manner.

The term "immune system cell" or "immune cell" refers to any cell of the immune system derived from hematopoietic stem cells in the bone marrow. Hematopoietic stem cells produce two major lineages, myeloid progenitor cells (producing myeloid cells, such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (producing lymphoid cells, such as T cells, B cells, and Natural Killer (NK) cells). Exemplary immune system cells include cd4+ T cells, cd8+ T cells, CD4-CD 8-double negative T cells, γδ T cells, regulatory T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells may also be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when the Major Histocompatibility Complex (MHC) receptor on the surface of APCs complexed with a peptide interacts with a TCR on the surface of the T cells.

The term "T cell" refers to a cell of the T cell lineage. By "cells of the T cell lineage" is meant cells that exhibit at least one phenotypic characteristic of a T cell or precursor or progenitor cell thereof, which distinguishes the cell from other lymphoid cells and cells of the erythroid or myeloid lineage. Such phenotypic characteristics may comprise expression of one or more proteins specific for T cells (e.g., CD3 ⁺、CD4⁺、CD8⁺), or physiological, morphological, functional or immunological characteristics specific for T cells. For example, cells of the T cell lineage can be progenitor or precursor cells committed to the T cell lineage; CD25 ⁺ immature and unactivated T cells; cells that have undergone CD4 or CD8 lineage targeting; CD4 ⁺CD8⁺ double positive thymus progenitor cells; single positive CD4 ⁺ or CD8 ⁺; tcrαβ or tcrγδ; or mature and functional or activated T cells. The term "T cell" encompasses naive T cells (cd45ra+, ccr7+, cd62l+, cd27+, CD45 RO-), central memory T cells (CD 45RO ⁺、CD62L⁺、CD8⁺), effector memory T cells (cd45ra+, CD45RO-, CCR7-, CD62L-, CD 27-), mucosa-associated invariant T (MAIT) cells, tregs, natural killer T cells and tissue resident T cells.

The term "B cell" refers to a cell of the B cell lineage. By "cells of the B cell lineage" is meant cells that exhibit at least one phenotypic characteristic of B cells or precursors or progenitors thereof, which distinguish the cells from other lymphoid cells and cells of the erythroid or myeloid lineage. Such phenotypic characteristics may comprise expression of one or more proteins specific for B cells (e.g., CD19 ⁺、CD72+、CD24+、CD20⁺), or physiological, morphological, functional or immunological characteristics specific for B cells. For example, the cells of the B cell lineage can be progenitor cells or precursor cells committed to the B cell lineage (e.g., pre-progenitor-B cells, and pre-B cells); immature and unactivated B cells or mature and functional or activated B cells. Thus, "B cells" encompass naive B cells, plasma cells, regulatory B cells, border zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablasts, and memory B cells (e.g., CD27 ⁺、IgD^-).

The term "cytotoxic activity", also referred to as "cytolytic activity", with respect to cells expressing on their surface an immune receptor (e.g., TCR) or chimeric Tim4 receptor (e.g., T cells or NK cells) according to the present disclosure, means that upon antigen-specific signaling (e.g., via TCR, chimeric Tim4 receptor), the cells induce the target cells to undergo apoptosis. In some embodiments, the cytotoxic cells may induce apoptosis in the target cells via release of cytotoxins, such as perforins, granzymes, and granulysins, from the particles. Perforin is inserted into the target cell membrane and forms pores that allow water and salts to quickly enter the target cell. Granzymes are serine proteases that induce apoptosis in target cells. Granulysin is also capable of forming pores in the target cell membrane and is a pro-inflammatory molecule. In some embodiments, cytotoxic cells can induce apoptosis in target cells via the interaction of Fas ligand with Fas molecules expressed on the target cells, which are up-regulated on T cells following antigen specific signaling. Fas is an apoptotic signaling receptor molecule on the surface of many different cells.

The term "depletion" in reference to an immune cell refers to a state of immune cell dysfunction, which is defined as a state of poor effector function (e.g., reduced cytokine production, reduced cytotoxic activity), reduced proliferative capacity, increased expression of an immunocheckpoint molecule, and transcription that is different from a functional effector or memory cell. In certain embodiments, the depleted immune cells become unresponsive to the presence of their target antigens. Immune cell depletion may be due to chronic exposure to the target antigen (e.g., possibly due to chronic infection) or when it enters an immunosuppressive environment (e.g., tumor microenvironment). In certain embodiments, immune cell depletion refers to T cell depletion, NK cell depletion, or both. In certain embodiments, the depleted T cells exhibit: (a) Increased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof; (b) Reduced production of IFN-gamma, IL-2, TNF-alpha, or any combination thereof; or both (a) and (b). In certain embodiments, the depleted NK cells exhibit: (a) Increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) reduced production of IFN- γ, TNF- α, or both; or both (a) and (b).

A "disease" is a state of health of a subject, wherein the subject is unable to maintain homeostasis, and wherein the subject's health continues to deteriorate if the disease is not improved. Conversely, a "disorder" or "undesired condition" of a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is not as good as in the absence of the disorder or undesired condition. The disorder or condition does not necessarily result in a further decrease in the health of the subject if not treated.

As used herein, the term "cancer" is defined as a disease characterized by rapid and uncontrolled growth of abnormal cells. Abnormal cells may form solid tumors or constitute hematological malignancies. Cancer cells can spread locally or through the blood stream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like.

The terms "subject," "patient," and "individual" are used interchangeably herein and are intended to encompass a living organism (e.g., a mammal) in which an immune response may be elicited. Examples of subjects include humans, primates, cattle, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, and transgenic species thereof.

"Adoptive cell immunotherapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy may be autologous (immune cells from the recipient), allogeneic (immune cells from a donor of the same species) or syngeneic (immune cells from a donor genetically identical to the recipient).

By "autologous" is meant any material (e.g., organ, tissue, graft of cells) from the same subject that is subsequently reintroduced into the subject.

"Allograft" refers to grafts from different subjects of the same species.

A "therapeutically effective amount" or "effective amount" of a chimeric protein or a cell expressing a chimeric protein (e.g., a chimeric Tim4 receptor or a cell expressing a chimeric Tim4 receptor) of the present disclosure refers to an amount of a protein or cell sufficient to result in an improvement in one or more symptoms of the disease, disorder, or undesired condition being treated. When referring to a single active ingredient administered alone or a cell expressing a single active ingredient, a therapeutically effective dose refers to the effect of the ingredient administered alone or the cell expressing the ingredient. When referring to a combination, a therapeutically effective dose refers to the combined amount of the active ingredients or combined co-active ingredients and the cells expressing the active ingredients that produce a therapeutic effect, whether administered sequentially or simultaneously.

"Treatment" or "amelioration" refers to the medical management of a disease, disorder, or undesired condition in a subject. Typically, the appropriate dose or treatment regimen comprising host cells expressing the chimeric proteins of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventative benefits include improved clinical outcome; alleviating or alleviating symptoms associated with a disease, disorder, or undesired condition; reducing the occurrence of symptoms; improving the quality of life; prolonging disease-free status; reducing the extent of a disease, disorder, or undesired condition; stabilizing the disease state; delay disease progression; relief; survival; prolonging the life cycle; or any combination thereof.

The term "anti-tumor effect" refers to a biological effect that may be manifested as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancer condition. "anti-tumor effects" may also be manifested by prevention of hematological malignancies or neoplasia.

An "autoimmune disease" refers to a condition caused by an autoimmune response. Autoimmune diseases are the result of an excessive response to self-antigens. The autoimmune response may involve autoreactive B cells, autoreactive T cells, or both that produce the autoantibody. As used herein, an "autoantibody" is an antibody produced by a subject that binds to an autoantigen also produced by the subject.

Additional definitions are provided throughout this disclosure.

Chimeric Tim4 receptors

The present disclosure provides chimeric Tim4 receptors comprising a single-chain chimeric protein comprising: (a) An extracellular domain comprising a binding domain, the binding domain comprising: a Tim4 IgV domain and a Tim4 mucin domain; (b) An intracellular signaling domain, wherein the intracellular signaling domain comprises an intracellular signaling domain comprising a signaling domain comprising an immune receptor tyrosine activation motif (ITAM), a costimulatory signaling domain, and a TLR signaling domain; and a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

In some embodiments, the chimeric Tim4 receptor comprises a single-chain chimeric protein comprising: (a) an extracellular domain comprising a Tim4 binding domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, a CD3 zeta signaling domain, and a TLR2 signaling domain; and (c) a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain. In certain embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17. In certain embodiments, the extracellular domain of a chimeric Tim4 receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain.

In some embodiments, the chimeric Tim4 receptor comprises a single chain chimeric protein comprising, from N-terminus to C-terminus: (a) an extracellular domain comprising a Tim4 binding domain; (b) a CD28 transmembrane domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, (c) a CD3 zeta signaling domain, and (d) a TLR2 signaling domain. In some embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17.

In some embodiments, the chimeric Tim4 receptor comprises a single chain chimeric protein comprising, from N-terminus to C-terminus: (a) an extracellular domain comprising a Tim4 binding domain; (b) a CD28 transmembrane domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, (c) a TLR2 signaling domain, and (d) a CD3 zeta signaling domain. In some embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence set forth in SEQ ID No. 17.

Additional chimeric Tim4 receptors are provided in the present disclosure.

In certain embodiments, the extracellular domain of a chimeric Tim4 receptor described herein optionally comprises an extracellular spacer domain located between and linking the binding domain and the transmembrane domain.

When expressed in a host cell, the chimeric Tim4 receptors of the present disclosure can confer T cell cytotoxicity specific for phosphatidylserine (e.g., the host cell becomes cytotoxic to stressed, damaged, injured, apoptotic, or necrotic cells expressing phosphatidylserine on its surface) to a modified host cell (e.g., a T cell) that has phagocytosis and innate functions of antigen processing and presentation. The intracellular CD28 signaling domain and CD3 zeta signaling domain act as T cell activators. Intracellular TLR2 signaling domains enhance the ability of host cells to present exogenous soluble antigens and activate class I restricted T cells. In some embodiments, the chimeric Tim4 receptor induces apoptosis in the targeted cells via release of granzyme, perforin, granulysin, or any combination thereof. In some embodiments, cells expressing a chimeric Tim4 receptor according to the present description exhibit a phagocytic phenotype specific for phosphatidylserine presenting cells. In some embodiments, cells (e.g., T cells) expressing a chimeric Tim4 receptor according to the disclosure exhibit enhanced antigen presenting activity. Without wishing to be bound by theory, the combination of T cell antigen capture and presentation capabilities with inducible and target-specific cytotoxic functions in single T cells suggests the potential for secondary immune responses via activation and enhancement of endogenous anti-tumor immunity.

T cells comprising a chimeric Tim4 receptor that includes from N-terminus to C-terminus: (a) an extracellular domain comprising a Tim4 binding domain; (b) a CD28 transmembrane domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, (c) a CD3 zeta signaling domain, and (d) a TLR2 TIR signaling domain, such as, for example, a chimeric Tim4 receptor comprising the amino acid sequence shown in SEQ ID No. 19. Further, from the N-terminus to the C-terminus, there are: (a) an extracellular domain comprising a Tim4 binding domain; (b) a CD28 transmembrane domain; (b) A chimeric Tim4 receptor comprising a CD28 signaling domain, (C) a CD3 zeta signaling domain, and (d) an intracellular signaling domain of a TLR2 TIR signaling domain (such as, for example, a chimeric Tim4 receptor comprising the amino acid sequence shown in SEQ ID NO: 19) and comprising, from N-terminus to C-terminus: (a) an extracellular domain comprising a Tim4 binding domain; (b) a CD28 transmembrane domain; (b) A chimeric Tim4 receptor comprising a CD28 signaling domain, (c) a TLR2 TIR signaling domain, and (d) an intracellular signaling domain of a CD3 zeta signaling domain (such as, for example, a chimeric Tim4 receptor comprising the amino acid sequence shown in SEQ ID NO: 18) exhibits more consistent antigen presenting function.

The intracellular signaling domain may comprise one or more effector domains capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the chimeric Tim4 receptor to phosphatidylserine. Signaling through the intracellular signaling domain is triggered by the binding of the extracellular domain to phosphatidylserine. The signal transduced by the intracellular signaling domain promotes effector function of cells containing the chimeric Tim4 receptor. Examples of effector functions include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, amplification, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen presentation, or any combination thereof.

In certain embodiments, the intracellular signaling domain comprises a first intracellular signaling domain. In embodiments, the intracellular signaling domain comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a first intracellular signaling domain, a second intracellular signaling domain, and a third intracellular signaling domain. Chimeric Tim4 receptors according to the present disclosure can be used in a variety of therapeutic methods, where scavenging apoptotic, necrotic, injured or stressed cells is beneficial, while providing co-stimulation that enhances cellular immune responses, reduces immune cell depletion, or both.

The components of the fusion proteins of the present disclosure are described in further detail herein.

Extracellular domain

As described herein, the chimeric Tim4 receptor includes an extracellular domain that includes a Tim4 binding domain. The Tim4 binding domain confers phosphatidylserine (PtdSer) specificity, a negatively charged headgroup phospholipid and is a component of the cell membrane. In healthy cells, phosphatidylserine is preferentially found in the inner leaflet of the cell membrane. However, when cells are stressed, damaged or undergo apoptosis or necrosis, phosphatidylserine is exposed on the outer leaflet of the cell membrane. Thus, phosphatidylserine can be used as a marker to distinguish stressed cells, damaged cells, apoptotic cells, necrotic cells, pyrophosphoric cells, or apoptotic cells. Binding of phosphatidylserine via the Tim binding domain can block interactions between phosphatidylserine and another molecule, and for example, interfere with, reduce, or eliminate certain functions of phosphatidylserine (e.g., signal transduction). In some embodiments, the binding of phosphatidylserine may induce certain biological pathways or recognize phosphatidylserine molecules or cells expressing phosphatidylserine for elimination.

The Tim4 binding domain of the chimeric Tim4 receptor suitable for use in the present disclosure can be any polypeptide or peptide derived from a Tim4 molecule that specifically binds phosphatidylserine. In embodiments, the Tim4 binding domain comprises an IgV domain from Tim4 and a mucin domain from Tim 4.

Phosphatidylserine binding is typically regulated by IgV domains. The core phosphatidylserine binding domain is a four amino acid sequence in the IgV domain (e.g., amino acids 95-98 of SEQ ID NO: 3). The Tim4 binding domain binds minimally to cells with low phosphatidylserine density.

In some embodiments, the Tim4 binding domain is obtained or derived from human Tim 4. Exemplary human Tim4 molecules are provided in Uniprot. Reference Q96H15 (SEQ ID NO: 1). Exemplary human Tim4 binding domains include or consist of the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6. Exemplary mouse Tim4 binding domains include or consist of the amino acid sequence of SEQ ID NO. 2 or amino acids 23-279 of SEQ ID NO. 2. In certain embodiments, the Tim4 binding domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 2, or amino acids 23-279 of SEQ ID NO 2. In certain embodiments, the Tim4 binding domain comprises an amino acid sequence having at least about 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 2, or amino acids 23-279 of SEQ ID NO. 2.

In some embodiments, the Tim4 binding domain comprises an IgV domain from Tim 4. An exemplary human Tim4IgV domain is provided in SEQ ID NO. 3. In some embodiments, the Tim4IgV domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID NO. 3. In certain embodiments, the Tim4IgV domain comprises an amino acid sequence having at least about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the Tim4 binding domain comprises a mucin domain from Tim 4. An exemplary human Tim4 mucin domain is provided in SEQ ID NO. 4. In certain embodiments, the Tim4 mucin domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID No. 4. In certain embodiments, the Tim4 mucin domain comprises an amino acid sequence having at least about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID No. 4.

In some embodiments, the Tim binding domain comprises a Tim4 IgV domain and a Tim4 mucin domain. In some embodiments, the Tim4 IgV domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 3 and the Tim4 mucin domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 4. In some embodiments, the Tim4 IgV domain and the Tim4 mucin domain together comprise or consist of the amino acid sequence set forth in SEQ ID NO. 5 or SEQ ID NO. 6. In some embodiments, the Tim4 binding domain or Tim4 IgV domain further comprises the Tim4 signal sequence of SEQ ID NO. 7.

In some embodiments, the extracellular domain optionally includes an extracellular non-signaling spacer or a linker domain. Such spacer or linker domains, if included, can position the binding domain at a location remote from the surface of the host cell to further achieve proper cell/cell contact, binding and activation. When included in a chimeric receptor as described herein, the extracellular spacer domain is typically located between the extracellular binding domain and the transmembrane domain of the chimeric Tim4 receptor. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, the selected binding epitope, the binding domain size, and affinity (see, e.g., guest et al, journal of immunotherapy (j. Immunother.), 28:203-11, 2005; pct publication No. WO 2014/031687). In some embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., igG1, igG2, igG3, igG4, igA, igD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. An altered IgG ₄ hinge region is described in PCT publication No. WO2014/031687, which is incorporated herein by reference in its entirety. In some embodiments, the extracellular spacer domain includes a modified IgG ₄ hinge region having the amino acid sequence of ESKYGPPCPPCP (SEQ ID NO: 9). Other examples of hinge regions that may be used for the chimeric Tim4 receptors described herein include hinge regions from extracellular regions of type 1 membrane proteins (e.g., CD8a, CD4, CD28, and CD 7), which may be wild-type or variants thereof. In some embodiments, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO. 10. In some embodiments, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT publication WO2014/031687, which is incorporated herein by reference in its entirety). In some embodiments, the extracellular spacer domain may include a stem region of a type II C-lectin (an extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins comprise CD23, CD69, CD72, CD94, NKG2A and NKG2D.

In some embodiments, the extracellular domain comprises an amino acid sequence from any mammalian species, including human, primate, cow, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, transgenic species thereof, or any combination thereof. In certain embodiments, the extracellular domain is murine, human, or chimeric.

Intracellular signaling domains

The intracellular signaling domain of the chimeric Tim4 receptor as described herein is an intracellular effector domain and is capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the chimeric Tim4 receptor to phosphatidylserine. The signal transduced by the intracellular signaling domain promotes effector function of cells containing the chimeric Tim4 receptor. Examples of effector functions include cytotoxic activity, secretion of cytokines, proliferation, anti-apoptotic signaling, persistence, amplification, phagocytosis of target cells or particles expressing phosphatidylserine on their surface, antigen capture, antigen processing, antigen presentation, or any combination thereof.

The intracellular signaling domain comprises a primary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, a secondary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain, a secondary intracellular signaling domain, and a tertiary intracellular signaling domain. The primary intracellular signaling domain, the secondary intracellular signaling domain, and/or the tertiary intracellular signaling domain may independently be any portion of a signaling molecule that retains sufficient signaling activity. In some embodiments, a full-length signaling molecule or a full-length intracellular component of a signaling molecule is used. In some embodiments, a truncated portion of the signaling molecule or an intracellular component of the signaling molecule is used, provided that the truncated portion retains sufficient signaling activity. In some embodiments, the signaling domain is a variant of a complete or truncated portion of a signaling molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

In some embodiments, the intracellular signaling domain comprises a CD28 signaling domain, a TLR2 signaling domain, and a CD3 zeta signaling domain. In some embodiments, the TLR2 signaling domain comprises a TLR2 TIR signaling domain. In some embodiments, the TLR2 TIR signaling domain is a TLR2 signaling domain having a HRFHGLWYMKMMWAWLQAKRKPRKAPSRN peptide sequence removed at the N-terminus of the TLR2 signaling domain.

As used herein, the designations of primary, secondary, and tertiary intracellular signaling domains include, but are not limited to, an arrangement of primary, intermediate, secondary, and tertiary intracellular signaling domains at the N-terminus of the intracellular portion of the chimeric Tim4 receptor. Thus, the naming of the primary intracellular signaling domain does not limit the use of the selected intracellular signaling domain at the N-terminus of the intracellular portion of the chimeric Tim4 receptor. The naming of the secondary intracellular signaling domains does not limit the use of the selected intracellular signaling domain in the middle of the intracellular portion of the chimeric Tim4 receptor (or at the C-terminus for those chimeric Tim4 receptors having only two intracellular signaling domains). The naming of tertiary intracellular signaling domains does not limit the use of selected intracellular signaling domains at the C-terminus of the intracellular portion of the chimeric Tim4 receptor. Thus, different arrangements of primary, secondary and/or tertiary intracellular signaling domains in the intracellular portion of the chimeric Tim4 receptor are contemplated.

Exemplary CD28 signaling domains include or consist of the amino acid sequence of SEQ ID NO. 12 or SEQ ID NO. 13. Exemplary CD3 zeta signaling domains include or consist of the amino acid sequence of SEQ ID NO. 14 or SEQ ID NO. 15. Exemplary TLR2 signaling domains include or consist of the amino acid sequence of SEQ ID NO. 16 or SEQ ID NO. 17.

In some embodiments, the CD28 signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, the CD28 signaling domain comprises or consists of the amino acid sequence set forth in SEQ ID NO. 13. In some embodiments, the CD3 zeta signaling domain comprises or consists of the amino acid sequence shown in SEQ ID NO. 14. In some embodiments, the CD3 zeta signaling domain comprises or consists of the amino acid sequence shown in SEQ ID NO. 15. In some embodiments, the TLR2 signaling domain comprises or consists of the amino acid sequence of SEQ ID NO. 16. In some embodiments, the TLR2 signaling domain is a TLR2 TIR signaling domain comprising or consisting of the amino acid sequence of SEQ ID No. 17.

In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus: a CD28 primary intracellular signaling domain, a TLR2 secondary intracellular signaling domain, and a cd3ζ tertiary intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus: a CD28 primary intracellular signaling domain, a CD3 zeta secondary intracellular signaling domain, and a TLR2 tertiary intracellular signaling domain. In some embodiments, the TLR2 intracellular signaling domain is a TLR2 TIR signaling domain.

In some embodiments, the intracellular signaling domain comprises a combination of a primary intracellular signaling domain, a secondary intracellular signaling domain, and a tertiary intracellular signaling domain as set forth in SEQ ID NO:39 or SEQ ID NO: 40.

The intracellular signaling domain may be from mammalian species, including humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, and transgenic species thereof.

Transmembrane domain

The transmembrane domain of the chimeric Tim4 receptor connects and is located between the extracellular domain and the intracellular signaling domain. The transmembrane domain is a hydrophobic alpha helix that passes through the host cell membrane. The transmembrane domain may be fused directly to the binding domain or extracellular spacer domain, if present. In certain embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptor, cluster of Differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, etc.). In one embodiment, the transmembrane domain is selected from the same molecules as the molecules from which the extracellular domain is derived. In another embodiment, the transmembrane domain is selected from the same molecule as the molecule from which the intracellular signaling domain is derived. For example, the chimeric Tim4 receptor can include a CD28 transmembrane domain and a CD28 costimulatory signaling domain. In certain embodiments, the transmembrane domain and the extracellular domain are from different molecules; the transmembrane domain and the intracellular signaling domain are from different molecules; or the transmembrane domain, extracellular domain and intracellular signaling domain are all from different molecules. In some embodiments, the transmembrane domain is a CD28 transmembrane domain. Exemplary CD28 transmembrane domains include or consist of the amino acid sequence of SEQ ID NO. 11. In certain embodiments, the transmembrane domain comprises or consists of an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity to SEQ ID No. 11. In certain embodiments, the transmembrane domain comprises an amino acid sequence having at least about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., deletions, additions, substitutions) to the amino acid sequence of SEQ ID NO. 11.

The transmembrane domain may be from any mammalian species, including human, primate, bovine, equine, caprine, ovine, canine, feline, murine, rat, rabbit, guinea pig, porcine, and transgenic species thereof.

In certain embodiments, the chimeric Tim4 receptor is encoded by a polynucleotide sequence from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, transgenic species thereof, or any combination thereof. In certain embodiments, the chimeric Tim4 receptor is murine, chimeric, human, or humanized.

It is to be understood that direct fusion of one domain of the chimeric Tim4 receptor described herein to another domain does not preclude the presence of an intermediate linking amino acid. The linking amino acids may be natural or unnatural (e.g., from the construct design of the chimeric protein). For example, the linking amino acids may be from restriction enzyme sites used to link one domain to another domain or to clone a polynucleotide encoding a chimeric Tim4 receptor into a vector.

Exemplary chimeric Tim4 receptors

Exemplary chimeric Tim4 receptors and component sequences are described in tables 1 and 2. In some embodiments, the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID NO:18 or the amino acid sequence of SEQ ID NO:18 lacking the signal sequence (amino acids 1-24). In some embodiments, the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID NO. 19 or the amino acid sequence of SEQ ID NO. 19 lacking the signal sequence (amino acids 1-24).

Table 1: chimeric Tim4 receptors and compositions

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In some embodiments, the chimeric Tim4 receptor comprises a Tim4 binding domain, a CD28 transmembrane domain, a CD28 primary intracellular signaling domain, a TLR2 TIR secondary intracellular signaling domain, and a CD3 zeta tertiary intracellular signaling domain. In some embodiments, the chimeric Tim4 receptor comprises a Tim4 binding domain comprising SEQ ID NO. 5, a CD28 transmembrane domain comprising SEQ ID NO. 11, a CD28 primary intracellular signaling domain comprising SEQ ID NO. 12, a TLR2 TIR secondary intracellular signaling domain comprising SEQ ID NO. 17, and a CD3 zeta tertiary intracellular signaling domain comprising SEQ ID NO. 14. In some embodiments, the chimeric Tim4 receptor further comprises the N-terminal signal peptide of SEQ ID NO. 7. In some embodiments, the chimeric Tim4 receptor comprises or consists of SEQ ID NO 18 or SEQ ID NO 18 lacking a signal peptide (amino acids 1-24).

In some embodiments, the chimeric Tim4 receptor comprises a Tim4 binding domain, a CD28 transmembrane domain, a CD28 primary intracellular signaling domain, a CD3 ζ secondary intracellular signaling domain, and a TLR2TIR tertiary intracellular signaling domain. In some embodiments, the chimeric Tim4 receptor comprises a Tim4 binding domain comprising SEQ ID NO. 5, a CD28 transmembrane domain comprising SEQ ID NO. 11, a CD28 primary intracellular signaling domain comprising SEQ ID NO. 12, a CD3 zeta secondary intracellular signaling domain comprising SEQ ID NO. 14, and a TLR2TIR tertiary intracellular signaling domain comprising SEQ ID NO. 17. In some embodiments, the chimeric Tim4 receptor further comprises the N-terminal signal peptide of SEQ ID NO. 7. In some embodiments, the chimeric Tim4 receptor comprises or consists of SEQ ID NO 19 or SEQ ID NO 19 lacking a signal peptide (amino acids 1-24).

Having specific domains and arrangements from N-terminal to C-terminal: the chimeric Tim4 receptor (as provided by the amino acid sequence shown in SEQ ID NO:19 or SEQ ID NO:19 lacking the signal peptide (amino acids 1-24)) of the Tim4 binding domain, CD28 transmembrane domain, CD28 primary intracellular signaling domain, CD3 zeta secondary intracellular signaling domain and TLR2 TIR tertiary intracellular signaling domain may have improved properties compared to a chimeric Tim4 receptor (as provided by the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:18 lacking the signal peptide (amino acids 1-24) (also referred to as CER 1234)) having the same domain but exchanged for the positions of the TLR2 TIR and CD3 zeta signaling domains. T cells transduced with chimeric phagocytic receptors according to SEQ ID NO 19 or SEQ ID NO 19 lacking a signal peptide (amino acids 1-24), also known as CER 1236T cells, show any combination of: (i) More consistent and higher inducibility in response to phosphatidylserine stimulation in multiple donor T cell samples; (ii) a more consistent antigen presentation function; (iii) Higher CD4 to CD 8T cell ratios in multiple donors compared to CER 1234T cells. In addition, T cells transduced with chimeric phagocytic receptors according to SEQ ID NO. 19 or SEQ ID NO. 19 lacking the signal peptide (amino acids 1-24) exhibit a major central memory and effector memory phenotype, repeated activation in response to successive phosphatidylserine exposures, and synergistic activity against target cells when combined with PARP inhibitors, BTK inhibitors or EGFR inhibitors.

Table 2: exemplary chimeric phagocytic receptors

Polynucleotides, vectors and host cells

The present disclosure provides nucleic acid molecules encoding any one or more of the chimeric Tim4 receptors described herein. Nucleic acid may refer to single-or double-stranded DNA, cDNA or RNA, and may comprise the positive and negative strands of nucleic acid complementary to each other, including antisense DNA, cDNA and RNA. The nucleic acid may be DNA or RNA in naturally occurring or synthetic form. Nucleic acid sequences encoding the desired chimeric Tim4 receptor can be obtained or generated by recombinant methods known in the art using standard techniques, such as by screening libraries from cells expressing the desired sequence or portions thereof, by deriving the sequence from vectors known to contain the sequence, or by isolating the sequence or portions thereof directly from cells or tissues containing the sequence, as described, for example, in Sambrook et al (1989 and 2001; molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press), new York), and Ausubel et al (molecular biology latest protocol (Current Protocols in Molecular Biology), 2003). Alternatively, the sequence of interest may be synthetically produced, rather than cloned.

The polynucleotide encoding the chimeric Tim4 receptor compositions provided herein can be from any animal, such as human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, or a combination thereof. In certain embodiments, the polynucleotide encoding the chimeric Tim4 receptor is from the same animal species as the host cell into which the polynucleotide is inserted.

Polynucleotides encoding the chimeric Tim4 receptors of the disclosure may be operably linked to expression control sequences. Expression control sequences may comprise appropriate transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; a sequence that stabilizes cytoplasmic mRNA; sequences that increase translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and sequences that may enhance protein secretion.

In certain embodiments, the polynucleotide encoding the chimeric Tim4 receptor includes a sequence encoding a signal peptide (also referred to as a leader peptide or signal sequence) at the 5' end for targeting the precursor protein to the secretory pathway. During cellular processing and localization of the chimeric Tim4 receptor on the host cell membrane, the signal peptide is optionally cleaved from the N-terminus of the extracellular domain. The polypeptide from which the signal peptide sequence is cleaved or removed may also be referred to as a mature polypeptide. Examples of signal peptides that can be used for the chimeric Tim4 receptor of the present disclosure include signal peptides derived from endogenous secreted proteins, including, for example, GM-CSF (amino acid sequence of SEQ ID NO: 27), tim1 (amino acid sequence of SEQ ID NO: 28), or Tim4 (amino acid sequence of SEQ ID NO:7 or 8). In certain embodiments, the polynucleotide sequence encodes a mature chimeric Tim4 receptor polypeptide, or the polypeptide sequence comprises a mature chimeric Tim4 receptor polypeptide. It will be appreciated by those skilled in the art that for the sequences disclosed herein comprising a signal peptide sequence, the signal peptide sequence may be replaced with another signal peptide capable of transporting the encoded protein to the extracellular membrane.

In certain embodiments, the chimeric Tim4 receptor encoding the polynucleotides of the present disclosure is codon optimized for efficient expression in a target host cell comprising the polynucleotide (see, e.g., scholten et al, clinical immunology (clin. Immunol.)), 119:135-145 (2006)). As used herein. "codon-optimized" polynucleotides include heterologous polynucleotides whose codons are modified with silent mutations corresponding to the abundance of tRNA in the host cell of interest.

According to any of the embodiments disclosed herein, a single polynucleotide molecule may encode one, two or more chimeric Tim4 receptors. Polynucleotides encoding more than one transgene may include sequences (e.g., IRES, viral 2A peptide) disposed between each gene for polycistronic expression.

Polynucleotides encoding at least two transgenes (e.g., chimeric Tim4 receptor and CAR) provided in the present disclosure can be used to make up a tandem expression cassette. Tandem expression cassette refers to a component of a vector nucleic acid comprising at least two transgenes under the control of, or operably linked to, the same set of regulatory sequences used to tandem or co-express the at least two transgenes. Regulatory sequences that can be used in the tandem expression cassettes of the present disclosure include suitable transcription initiation sequences, termination sequences, promoter sequences, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; a sequence that stabilizes cytoplasmic mRNA; sequences that increase translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; a sequence that enhances protein secretion, or any combination thereof.

In one aspect, the present disclosure provides a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim4 receptor of the present disclosure and a polynucleotide encoding a cellular immunotherapeutic agent (e.g., CAR, TCR, etc.).

In certain embodiments, tandem expression cassettes can be constructed to optimize spatial and temporal control. For example, a tandem expression cassette may contain promoter elements to optimize spatial and temporal control. In some embodiments, the tandem expression cassette comprises a tissue-specific promoter or enhancer that is capable of specifically inducing the tandem expression cassette to an organ, cell type (e.g., immune cell), or pathological microenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can act synergistically or independently to activate transcription. In certain embodiments, the tandem expression cassette comprises a constitutive promoter. An exemplary constitutive promoter for the tandem expression cassette of the present disclosure is the EF-1. Alpha. Promoter. In certain embodiments, the tandem expression cassette comprises an inducible promoter. In certain embodiments, the tandem expression cassette comprises a tissue specific promoter.

The at least two transgenes contained in the tandem expression cassette may be in any order. For example, a tandem expression cassette comprising a polynucleotide encoding a chimeric Tim4 receptor and a polynucleotide encoding a CAR may be arranged from 5 'to 3' as: chimeric Tim4 receptor-CAR or CAR-chimeric Tim4 receptor.

In certain embodiments, a receptor comprising two or more polypeptide chains that associate to form a multimer or complex may be encoded by two or more polynucleotide molecules in a tandem expression construct. Exemplary multimeric receptors contemplated for expression in the tandem expression constructs of the present disclosure include the multiplex CAR, TCR, TCR-CAR and TRuC ^TM constructs. Thus, exemplary tandem expression cassette embodiments encoding a chimeric Tim4 receptor and a TCR may include polynucleotides encoding the chimeric Tim4 receptor, polynucleotides encoding a TCR alpha chain polypeptide, and polynucleotides encoding a TCR beta chain polypeptide.

In certain embodiments, the tandem expression cassettes of the present disclosure may include an Internal Ribosome Entry Site (IRES) or a peptide cleavage site, such as a furin cleavage site or a viral 2A peptide, disposed between each polynucleotide contained within the tandem expression cassette, to allow co-expression of multiple proteins from a single mRNA. For example, an IRES, furin cleavage site, or viral 2A peptide can be disposed within the tandem expression cassette between a polynucleotide encoding a chimeric Tim4 receptor and a polynucleotide encoding a CAR. In another example, an IRES, furin cleavage site, or viral 2A peptide may be disposed between a polynucleotide encoding a chimeric Tim4 receptor, a polynucleotide encoding a TCR alpha chain polypeptide, and a polynucleotide encoding a TCR beta chain polypeptide. In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), thosea asigna virus (T2A), equine rhinitis a virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. Exemplary T2A peptides include the amino acid sequence of any one of SEQ ID NOs 29-33. Exemplary P2A peptides include the amino acid sequence of SEQ ID NO 34 or 35. Exemplary E2A peptide sequences include the amino acid sequence of SEQ ID NO. 36. Exemplary F2A peptide sequences include the amino acid sequence of SEQ ID NO. 37.

Certain embodiments of the tandem expression cassettes of the present disclosure include polynucleotides encoding CARs/or TCRs that are specific for a target antigen (e.g., a tumor antigen) and polynucleotides encoding chimeric Tim4 receptors of the present disclosure. When target cells expressing a target antigen are bound by the CAR and/or TCR, cells modified to express such tandem expression cassettes induce apoptosis of the target cells. Apoptosis induces exposure of a pro-phagocytic marker (such as phosphatidylserine) on target cells, which can then target damaged or apoptotic cells for phagocytosis by the chimeric Tim4 receptor.

Polynucleotides encoding the desired chimeric Tim4 receptor can be inserted into suitable vectors, such as viral vectors, non-viral plasmid vectors, and non-viral vectors, e.g., lipid-based DNA vectors, modified mRNA (modRNA), self-amplified mRNA, CELiD, and transposon-mediated gene transfer (PiggyBac), for introduction into host cells of interest (e.g., immune cells). Polynucleotides encoding the chimeric Tim4 receptors of the present disclosure can be cloned into any suitable vector, such as an expression vector, a replication vector, a probe-generating vector, or a sequencing vector. In certain embodiments, the polynucleotide encoding the extracellular domain, the polynucleotide encoding the transmembrane domain, and the polynucleotide encoding the intracellular signaling domain are ligated together into a single polynucleotide, and then inserted into a vector. In other embodiments, the polynucleotide encoding the extracellular domain, the polynucleotide encoding the transmembrane domain, and the polynucleotide encoding the intracellular signaling domain, respectively, may be inserted into a vector such that the expressed amino acid sequence results in a functional chimeric Tim4 receptor. The vector encoding the chimeric Tim4 receptor is referred to herein as a "chimeric Tim4 receptor vector".

In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim4 receptor. In certain embodiments, the vector comprises a polynucleotide encoding two or more chimeric Tim4 receptors. In certain embodiments, a single polynucleotide encoding two or more chimeric Tim4 receptors is cloned into a cloning site and expressed from a single promoter, wherein each chimeric Tim4 receptor sequence is separated from each other by an Internal Ribosome Entry Site (IRES), furin cleavage site, or viral 2A peptide, to allow co-expression of multiple genes from a single open reading frame (e.g., a polycistronic vector). In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), thosea asigna virus (T2A), equine rhinitis a virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. Exemplary T2A peptides include the amino acid sequence of any one of SEQ ID NOs 29-33. Exemplary P2A peptides include the amino acid sequence of SEQ ID NO 34 or 35. Exemplary E2A peptide sequences include the amino acid sequence of SEQ ID NO. 36. Exemplary F2A peptide sequences include the amino acid sequence of SEQ ID NO. 37.

In certain embodiments, the vector comprises two or more polynucleotides, each polynucleotide encoding a chimeric Tim4 receptor. Two or more polynucleotides encoding chimeric Tim4 receptors may be cloned sequentially into a vector at different cloning sites, wherein each chimeric Tim4 receptor is expressed under the control of a different promoter. In certain embodiments, vectors are used that allow for long-term integration and propagation of transgenes to daughter cells. Examples include viral vectors, such as adenovirus, adeno-associated virus, vaccinia virus, herpes virus, cytomegalovirus, poxvirus, or retroviral vectors, such as lentiviral vectors. Lentiviral derived vectors can be used to achieve long term gene transfer and have additional advantages over vectors, including the ability to transduce non-proliferating cells (e.g., hepatocytes) and low immunogenicity.

In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim4 receptor and a polynucleotide encoding a cellular immunotherapeutic agent (e.g., chimeric antigen receptor, recombinant TCR, etc.). In certain embodiments, a single polynucleotide encoding a chimeric Tim4 receptor and a cellular immunotherapeutic (e.g., CAR) is cloned into a cloning site and expressed from a single promoter, wherein the chimeric Tim4 receptor sequence and the cellular immunotherapeutic (e.g., CAR) sequence are separated from each other by an Internal Ribosome Entry Site (IRES), a furin cleavage site, or a viral 2A peptide, to allow co-expression of multiple genes from a single open reading frame (e.g., a polycistronic vector). In certain embodiments, the viral 2A peptide is porcine teschovirus-1 (P2A), thosea asigna virus (T2A), equine rhinitis a virus (E2A), foot-and-mouth disease virus (F2A), or a variant thereof. Exemplary T2A peptides include the amino acid sequences of SEQ ID NOS.29-33. Exemplary P2A peptides include the amino acid sequence of SEQ ID NO 34 or 35. Exemplary E2A peptide sequences include the amino acid sequence of SEQ ID NO. 36. Exemplary F2A peptide sequences include the amino acid sequence of SEQ ID NO. 37.

In certain embodiments, a polynucleotide encoding a chimeric Tim4 receptor and a polynucleotide encoding a cellular immunotherapeutic (e.g., CAR) binding protein are ligated together into a single polynucleotide, and then inserted into a vector. In other embodiments, the polynucleotide encoding the CER and the polynucleotide encoding the CAR or TCR binding protein, respectively, may be inserted into the vector at the same or different cloning site such that the expressed amino acid sequence produces a functional CER and CAR/or TCR. Vectors encoding tandem expression cassettes are referred to herein as "tandem expression vectors".

In certain embodiments, the vector comprises a polynucleotide encoding a chimeric Tim4 receptor and a polynucleotide encoding a cellular immunotherapeutic agent (e.g., CAR). Polynucleotides encoding the chimeric Tim4 receptor and the cellular immunotherapeutic (e.g., CAR) may be cloned sequentially into vectors at different cloning sites, with the chimeric Tim4 receptor and the cellular immunotherapeutic (e.g., CAR) expressed under the control of different promoters.

The vector encoding the core virus is referred to herein as a "viral vector". There are a number of viral vectors available for use in the compositions of the present disclosure, including those identified for use in human gene therapy applications (see Pfeifer and Verme, annual reviews of genomics and human genetics (an. Rev. Genomics hum. Genet.)) 2:177, 2001. Suitable viral vectors include RNA virus-based vectors, such as retroviral derived vectors, e.g., a maroney Murine Leukemia Virus (MLV) derived vector, and more complex retroviral derived vectors, e.g., lentiviral derived vectors. HIV-1 derived vectors belong to this class. Other examples include lentiviral vectors from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (sheep lentivirus). Methods for transducing mammalian host cells with viral particles containing chimeric receptor transgenes using retroviral and lentiviral vectors and packaging cells are known in the art and have been previously described, for example, in U.S. patent 8,119,772; walchli et al, public science library, complex (PLoS One) 6:327930, 2011; zhao et al, J.Immunol.) (174:4415, 2005; engels et al, human gene therapy (hum. Gene Ther.) 14:1155, 2003; frecha et al, molecular therapy (mol. Ther.) 18:1748, 2010; verhoeyen et al, methods of molecular biology (Methods mol. Biol.) 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

In certain embodiments, the viral vector is used to introduce a non-endogenous polynucleotide encoding a chimeric Tim4 receptor into a host cell. The viral vector may be a retroviral vector or a lentiviral vector. Viral vectors may also comprise nucleic acid sequences encoding markers for transduction. Transduction markers for viral vectors are known in the art and comprise selectable markers that may confer drug resistance, or detectable markers, such as fluorescent markers or cell surface proteins that may be detected by methods such as flow cytometry. In particular embodiments, the viral vector further comprises a gene marker for transduction comprising a fluorescent protein (e.g., green, yellow), an extracellular domain of human CD2, or truncated human EGFR (EGFR t or tEGFR; see Wang et al, blood (Blood) 118:1255, 2011). Exemplary tEGFR includes the amino acid sequence of SEQ ID NO: 38. When the viral vector genome comprises multiple genes to be expressed in a host cell as independent proteins from a single transcript, the viral vector may also comprise additional sequences between the two (or more) genes that allow for polycistronic expression. Examples of such sequences for viral vectors include an Internal Ribosome Entry Site (IRES), a furin cleavage site, a viral 2A peptide (e.g., T2A, P2A, E2A, F a), or any combination thereof.

Other viral vectors may also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV) -based vectors; vectors derived from Herpes Simplex Virus (HSV) include amplicon vectors, replication defective HSV and attenuated HSV (Krisky et al, gene therapy 5:1517, 1998).

Other viral vectors recently developed for gene therapy use may also be used with the compositions and methods of the present disclosure. Such vectors include vectors derived from baculovirus and alpha-virus. (Jolly, D J.1999, emerging viral Vectors (EMERGING VIRAL Vectors), pages 209-40, friedmann T. Edit, development of human gene therapy (The Development of Human GENE THERAPY), new York: cold spring harbor laboratory (New York: cold Spring Harbor Lab)), or plasmid Vectors (e.g., sleep bed or other transposon Vectors).

In certain embodiments, chimeric Tim4 receptor vectors can be constructed to optimize spatial and temporal control. For example, chimeric Tim4 receptor vectors can contain promoter elements to optimize spatial and temporal control. In some embodiments, the chimeric Tim4 receptor vector comprises a tissue-specific promoter or enhancer capable of specifically inducing the chimeric Tim4 receptor to an organ, cell type (e.g., immune cell), or pathological microenvironment, such as a tumor or infected tissue. An "enhancer" is an additional promoter element that can act synergistically or independently to activate transcription. In certain embodiments, the chimeric Tim4 receptor vector comprises a constitutive promoter. In certain embodiments, the chimeric Tim4 receptor vector comprises an inducible promoter. In certain embodiments, the chimeric Tim4 receptor vector comprises a tissue-specific promoter.

In certain embodiments, the chimeric Tim4 receptor vector can comprise a gene encoding a homing receptor, such as CCR4 or CXCR4, to enhance homing and anti-tumor activity in vivo.

Where time control is required, the chimeric Tim4 receptor vector may contain elements that allow for inducible elimination of the transduced cells. For example, such a vector may comprise an inducible suicide gene. Suicide genes may be apoptotic genes or genes that confer sensitivity to agents (e.g., drugs). Exemplary suicide genes include chemically inducible cysteine protease 9 (iCASP) (U.S. patent publication No. 2013/007434), chemically inducible Fas, or herpes simplex virus thymidine kinase (HSV-TK), which confers sensitivity to ganciclovir. In further embodiments, the chimeric Tim4 receptor vector can be designed to express a known cell surface antigen that is capable of clearing transduced cells upon infusion of the relevant antibody. Examples of cell surface antigens and their related antibodies that can be used to remove transduced cells include CD20 and rituximab, RQR8 (combined CD34 and CD20 epitopes, allowing for CD34 selection and anti-CD 20 deletion) and rituximab, as well as EGFR and cetuximab.

Inducible vector systems, such as the tetracycline (Tet) -On vector system (Heinz et al, human Gene therapy, 2011, 22:166-76) that activates transgene expression with doxycycline, may also be used for inducible chimeric Tim4 receptor expression. Inducible chimeric Tim4 receptor expression can also be achieved through the use of the RUSH (regeneration using A SELECTIVE hook) system based on streptavidin anchored to the endoplasmic reticulum membrane and streptavidin binding protein incorporated into the chimeric Tim4 receptor structure, wherein the addition of biotin to the system results in release of the chimeric Tim4 receptor from the endoplasmic reticulum (Agaugue et al 2015, molecular therapy 23 (supplement 1): S88).

In certain embodiments, host cells modified with a chimeric Tim4 receptor may also be modified to co-express one or more small gtpases. Rho GTPase, a small (about 21k Da) subfamily of signaling G proteins and Ras superfamily, regulates actin cytoskeletal organization in various cell types and promotes pseudopodia extension and phagosome closure during phagocytosis (see, e.g., castellano et al, 2000, journal of cytosciences (j. Cell sci.) 113:2955-2961). Phagocytosis requires the recruitment of F-actin under tethered cells or particles, and F-actin rearrangement to allow membrane extension, leading to cell or particle internalization. RhoGTPase comprises RhoA, rac1, rac2, rhoG and CDC42. Other small gtpases, such as Rap1, are involved in regulating complement-mediated phagocytosis. Co-expression of small GTPases with chimeric Tim4 receptors may promote internalization and/or phagosome formation of target cells or particles by host cells. In some embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on a different vector than the vector containing the chimeric Tim4 receptor. In other embodiments, the recombinant nucleic acid molecule encoding the GTPase is encoded on the same vector as the chimeric Tim4 receptor. The GTPase and chimeric Tim4 receptors can be expressed on the same vector under the control of different promoters (e.g., at different multiple cloning sites). Alternatively, the chimeric Tim4 receptor and GTPase may be expressed in a polycistronic vector under the control of one promoter. The polynucleotide sequence encoding the chimeric Tim4 receptor and the polynucleotide sequence encoding the small GTPase may be isolated from each other by IRES or viral 2A peptide in a polycistronic vector. Exemplary 2A peptides include T2A (SEQ ID NOS: 29-33), P2A (SEQ ID NOS: 34 or 35), E2A (SEQ ID NO: 36), F2A (SEQ ID NO: 37). Examples of gtpases that may be co-expressed with the chimeric Tim4 receptor include Rac1, rac2, rab5 (also known as Rab5 a), rab7, rap1, rhoA, rhoG, CDC42, or any combination thereof. In particular embodiments, the GTPase comprises or is a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the Rac1 amino acid sequence of SEQ ID NO:41, the Rab5 amino acid sequence of SEQ ID NO:42, the Rab7 amino acid sequence of SEQ ID NO:43, the Rap1 amino acid sequence of SEQ ID NO:44, the RhoA amino acid sequence of SEQ ID NO:45, the CDC42 amino acid sequence of SEQ ID NO:46, or any combination thereof.

In certain embodiments, a cell (e.g., an immune cell) obtained from a subject can be engineered into a non-natural or recombinant cell (e.g., a non-natural or recombinant immune cell) by introducing a polynucleotide encoding a chimeric Tim4 receptor as described herein, whereby the cell expresses a cell surface-localized chimeric Tim4 receptor. In certain embodiments, the host cell is an immune cell, such as a myeloid progenitor cell or lymphoid progenitor cell. Exemplary immune cells that can be modified to include a polynucleotide encoding a chimeric Tim4 receptor or a vector including a polynucleotide encoding a chimeric Tim4 receptor include T cells, natural killer cells, B cells, lymphoid precursor cells, antigen presenting cells, dendritic cells, langerhans cells, bone marrow precursor cells, mature bone marrow cells, monocytes or macrophages.

In certain embodiments, the B cells are genetically modified to express one or more chimeric Tim4 receptors. B cells have certain properties that may be advantageous as host cells, including: trafficking to the site of inflammation, internalization and presentation of antigen, co-stimulation of T cells, high proliferation and self-renewal (life-long duration). In certain embodiments, B cells modified with chimeric Tim4 receptors are capable of digesting phagocytic target cells or phagocytic target particles into smaller peptides and presenting them to T cells via MHC molecules. Antigen presentation by B cells modified with chimeric Tim4 receptors may aid in antigen diffusion of immune responses against non-targeted antigens. B cells include progenitor or precursor cells committed to the B cell lineage (e.g., pre-progenitor-B cells, and pre-B cells); immature and unactivated B cells; or mature and functional or activated B cells. In certain embodiments, the B cell may be a primary B cell, a plasma cell, a regulatory B cell, a border region B cell, a follicular B cell, a lymphoplasmacytoid cell, a plasmablast cell, a memory B cell, or any combination thereof. Memory B cells can be distinguished from primary B cells based on the lack of expression of CD27 on the primary B cells. In certain embodiments, the B cells may be primary cells or cell lines derived from humans, mice, rats, or other mammals. B cell lines are well known in the art. If obtained from a mammal, the B cells may be obtained from a variety of sources, including blood, bone marrow, spleen, lymph nodes or other tissues or fluids. The B cell composition may be enriched or purified.

In certain embodiments, the T cells are genetically modified to express one or more chimeric Tim4 receptors. Exemplary T cells include CD4 ⁺ helper cells, CD8 ⁺ effector cells (cytotoxicity), naive cells (cd45ra+, CCR7+, cd62l+, cd27+, CD45 RO-), central memory cells (CD 45RO ⁺、CD62L⁺、CD8⁺), effector memory cells (cd45ra+, CD45RO-, CCR7-, CD62L-, CD 27-), T memory stem cells, regulatory cells, mucosa-associated invariant cells (MAIT), γδ (gd), tissue resident T cells, natural killer T cells, or any combination thereof. In certain embodiments, the T cell may be a primary cell or cell line derived from a human, mouse, rat, or other mammal. If obtained from a mammal, T cells may be obtained from a variety of sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cell compositions can be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al, leukemia (Leukemia), 21:230, 2000. In certain embodiments, the T cell lacks endogenous expression of the tcra gene, the tcrp gene, or both. Such T cells may naturally lack endogenous expression of TCR a and β chains, or may have been modified to block expression (e.g., T cells from transgenic mice that do not express TCR a and β chains or cells that have been manipulated to inhibit expression of TCR a and β chains) or to knock out TCR a chains, TCR β chains, or both genes.

In certain embodiments, the host cell expressing the chimeric Tim proteins of the disclosure on the cell surface is not a T cell or a cell of the T cell lineage, but a progenitor cell, stem cell, or a cell modified to express cell surface anti-CD 3.

In certain embodiments, host cells modified with chimeric Tim4 receptors can also be modified to co-express a cellular immunotherapeutic (e.g., CAR, TCR, etc.). In some embodiments, the cellular immunotherapeutic agent comprises a Chimeric Antigen Receptor (CAR). CARs are recombinant receptors, which generally include: an extracellular domain comprising a binding domain that binds to a target antigen; an intracellular signaling domain (e.g., including an intracellular signaling domain comprising ITAM and optionally an intracellular co-stimulatory domain), and a transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

Binding domains suitable for use in the CARs of the present disclosure comprise any antigen binding polypeptide. The binding domain may comprise an antibody or antigen-binding fragment thereof, including, for example, full-length heavy chains, fab fragments, fab ', F (ab') ₂, sFv, VH domain, VL domain, dAb, VHH, CDR, and scFv. In certain embodiments, the CAR binding domain is murine, chimeric, human, or humanized.

In certain embodiments, the binding domain of the CAR targets a cancer or tumor antigen. Exemplary antigens that a CAR may target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79B, mesothelin 、PSMA、BCMA、ROR1、MUC-16、L1CAM、CD22、CD19、CD20、CD23、CD24、CD37、CD30、CA125、CD56、c-Met、EGFR、GD-3、HPV E6、HPV E7、MUC-1、HER2、 folate receptor alpha, CD97, CD171, CD179a, CD44v6, WT1, VEGF-alpha, VEGFR1, IL-13 ra 2, IL-11 ra, PSA, fcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, lewis a antigen, lewis Y antigen, MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD 2, and GD2.

In certain embodiments, the extracellular domain of a CAR provided in the present disclosure optionally includes an extracellular non-signaling spacer or linker domain. Where included, such a spacer or linker domain may position the binding domain at a location remote from the surface of the host cell to further allow for proper cell-to-cell contact, binding and activation. The extracellular spacer domain is typically located between the extracellular binding domain and the transmembrane domain of the CAR. The length of the extracellular spacer can be varied to optimize target molecule binding based on the selected target molecule, the selected binding epitope, the binding domain size, and affinity (see, e.g., guest et al, journal of immunotherapy, 28:203-11, 2005; pct publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., igG1, igG2, igG3, igG4, igA, igD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. An altered IgG ₄ hinge region is described in PCT publication No. WO 2014/031687, which is incorporated herein by reference in its entirety. In a particular embodiment, the extracellular spacer domain comprises a modified IgG ₄ hinge region having the amino acid sequence of SEQ ID NO. 9.

Other examples of hinge regions that can be used in the CARs described herein include hinge regions from extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28, and CD7, which can be wild-type or variants thereof. In a particular embodiment, the extracellular spacer domain comprises a CD8a hinge region having the amino acid sequence of SEQ ID NO: 172. In another specific embodiment, the extracellular spacer domain comprises a CD28 hinge region having the amino acid sequence of SEQ ID NO. 10. In further embodiments, the extracellular spacer domain comprises all or part of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT publication WO2014/031687, which is incorporated herein by reference in its entirety). In still further embodiments, the extracellular spacer domain may include a stem region of a type II C-lectin (an extracellular domain located between the C-lectin domain and the transmembrane domain). Type II C-lectins comprise CD23, CD69, CD72, CD94, NKG2A and NKG2D.

The CARs of the disclosure include a transmembrane domain connecting and between an extracellular domain and an intracellular signaling domain. The transmembrane domain ranges from about 15 amino acids to about 30 amino acids in length. The transmembrane domain is a hydrophobic alpha helix that passes through the host cell membrane and anchors the CAR in the host cell membrane. The transmembrane domain may be fused directly to the binding domain or extracellular spacer domain, if present. In certain embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptor, cluster of Differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, etc.). The transmembrane domain may be selected from the same molecule as the extracellular domain or intracellular signaling domain (e.g., the CAR comprises a CD28 costimulatory signaling domain and a CD28 transmembrane domain). In certain embodiments, the transmembrane domain and extracellular domain are each selected from different molecules. In other embodiments, the transmembrane domain and intracellular signaling domain are each selected from different molecules. In yet other embodiments, the transmembrane domain, extracellular domain and intracellular signaling domain are each selected from different molecules.

Exemplary transmembrane domains for the CARs of the present disclosure comprise CD28、CD2、CD4、CD8a、CD5、CD3ε、CD3δ、CD3ζ、CD9、CD16、CD22、CD25、CD27、CD33、CD37、CD40、CD45、CD64、CD79A、CD79B、CD80、CD86、CD95(Fas)、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD154(CD40L)、CD200R、CD223(LAG3)、CD270(HVEM)、CD272(BTLA)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、CD279(PD-1)、CD300、CD357(GITR)、A2aR、DAP10、FcRα、FcRβ、FcRγ、Fyn、GAL9、KIR、Lck、LAT、LRP、NKG2D、NOTCH1、NOTCH2、NOTCH3、NOTCH4、PTCH2、ROR2、Ryk、Slp76、SIRPα、pTα、TCRα、TCRβ、TIM3、TRIM、LPA5 and Zap70 transmembrane domains. Exemplary CD28 transmembrane domains include the amino acid sequence of SEQ ID NO. 11. In a particular embodiment, the transmembrane domain comprises a CD8a transmembrane domain having the amino acid sequence of SEQ ID NO: 174.

The intracellular signaling domain of the CAR is an intracellular effector domain and is capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the CAR to a target molecule (e.g., a cancer antigen) and activating at least one of a normal effector function or response of an immune cell (e.g., a T cell engineered to express the CAR). In some embodiments, the CAR induces a function of the T cell, such as cytolytic activity or T helper cell activity, such as secretion of cytokines or other factors. An intracellular signaling domain may be any portion of an intracellular signaling molecule that retains sufficient signaling activity. In some embodiments, the intracellular signaling domain is obtained from an antigen receptor component (e.g., TCR) or a co-stimulatory molecule. In some embodiments, the full-length intracellular signaling domain of an antigen receptor or co-stimulatory molecule is used. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor or co-stimulatory molecule is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the intracellular signaling domain is a variant of the full length or truncated portion of the intracellular signaling domain of the antigen receptor co-stimulatory molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

In some embodiments, the intracellular signaling domain of the CAR comprises a signaling domain comprising an immune receptor tyrosine activation motif (ITAM). The signaling domain containing ITAMs typically contains at least one (one, two, three, four or more) ITAM, which refers to a conserved motif of YXXL/I-X _6-8 -YXXL/I. The signaling domain containing ITAM can initiate T cell activation signaling following antigen binding or ligand binding. The ITAM signaling domain comprises intracellular signaling domains such as cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP12, fcrγ, and CD66 d. Exemplary CD3 zeta signaling domains that can be used in the CARs of the present disclosure include the amino acid sequences of SEQ ID NO:177 or SEQ ID NO: 178.

The CAR intracellular signaling domain optionally includes a co-stimulatory signaling domain that, when activated with a primary or classical (e.g., ITAM-driven) activation signal, promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. The co-stimulatory signaling domain for the CAR comprises, for example CD27、CD28、CD40L、GITR、NKG2C、CARD1、CD2、CD7、CD27、CD30、CD40、CD54(ICAM)、CD83、CD134(OX-40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD226、CD270(HVEM)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、DAP10、LAT、LFA-1、LIGHT、NKG2C、SLP76、TRIM、ZAP70 or any combination thereof. In some embodiments, the costimulatory signaling domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), or 4-1BB (CD 137) signaling domain. Exemplary CD28 costimulatory signaling domains that can be used in the CARs of the present disclosure include the amino acid sequences of SEQ ID NO:179 or 180. An exemplary 4-1BB costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 181. In certain embodiments, the CAR comprises one, two, or more co-stimulatory signaling domains.

In some embodiments, the CAR is a recombinant receptor consisting of an scFv binding domain derived from an antibody, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is from a TCR.

In certain embodiments, the chimeric antigen receptor comprises an amino acid sequence from any mammalian species, including human, primate, cow, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig, transgenic species thereof, or any combination thereof. In certain embodiments, the chimeric antigen receptor is murine, chimeric, human or humanized.

In certain embodiments, the CAR is a first generation CAR, a second generation CAR, or a third generation CAR. First generation CARs typically have an intracellular signaling domain comprising cd3ζ, fcγri, or other intracellular signaling domain containing an activation domain of ITAM to provide a T cell activation signal. The second generation CAR further comprises a costimulatory signaling domain (e.g., from an endogenous T cell costimulatory receptor, such as CD28, 4-1BB, or ICOS). The third generation CAR includes an activation domain comprising ITAM, a first costimulatory signaling domain, and a second costimulatory signaling domain.

In some embodiments, one or more of the extracellular domain, the binding domain, the linker, the transmembrane domain, the intracellular signaling domain, or the costimulatory domain comprises a linking amino acid. "connecting amino acid" or "connecting amino acid residues" refers to one or more (e.g., about 2-20) amino acid residues between two adjacent domains, motifs, regions, modules or fragments of a protein, such as between a binding domain and an adjacent linker, between a transmembrane domain and an adjacent extracellular or intracellular domain, or at one or both ends of a linker connecting the two domains, motifs, regions, modules or fragments (e.g., between a linker and an adjacent binding domain, or between a linker and an adjacent hinge). The linking amino acids may be created by the construct design of the fusion protein (e.g., amino acid residues generated during construction of the polynucleotide encoding the fusion protein by use of restriction sites or self-cleaving peptide sequences). For example, the transmembrane domain of the fusion protein may have one or more linking amino acids at the amino terminus, the carboxy terminus, or both.

In certain embodiments, the engineered host cell co-expresses the chimeric Tim4 receptor and the anti-CD 72 CAR.

In certain embodiments, the host cell modified with the chimeric Tim4 receptor co-expresses a recombinant TCR. Recombinant TCR proteins comprise "traditional" TCRs consisting of heterodimers of an alpha chain polypeptide and a beta chain polypeptide or heterodimers of a gamma chain polypeptide and a delta chain polypeptide, binding fragments thereof, and fusion proteins, including, for example, single chain TCRs, single domain TCRs, soluble TCR fusion TCR proteins, and TCR fusion constructs (TRuC ^TM). In certain embodiments, the tandem expression cassette includes a polynucleotide encoding a recombinant tcrp chain comprising a tcrp variable region and a tcrp constant region, and a polynucleotide encoding a recombinant tcra chain comprising a tcra variable region and a tcra constant region. In certain embodiments, the recombinant TCR is an enhanced affinity TCR. In one embodiment, the recombinant TCR is an enhanced affinity TCR.

In certain embodiments, the recombinant TCR-binding protein is a single chain TCR (scTCR) comprising vα linked to vβ via a flexible linker. In some embodiments, the scTCR comprises a vα -linker-vβ polypeptide. In other embodiments, the scTCR comprises a vβ -linker-vα polypeptide.

In certain embodiments, host cells modified with chimeric Tim4 receptors may also be modified to co-express single chain TCR (scTCR) fusion proteins. The scTCR fusion protein includes a binding domain comprising a scTCR (TCR V.alpha.domain linked to a TCR V.beta.domain), optionally an extracellular spacer, a transmembrane domain, and an intracellular component comprising a single intracellular signaling domain providing T cell activation signals (e.g., an activation domain containing CD3 zeta ITAM) and optionally a costimulatory signaling domain (see, aggen et al 2012, gene therapy 19:365-374; stone et al, cancer immunology and immunotherapy (Cancer immunology), 2014, 63:1163-76).

In certain embodiments, host cells modified with a chimeric Tim4 receptor can also be modified to co-express a T cell receptor-based chimeric antigen receptor (TCR-CAR). TCR-CARs are heterodimeric fusion proteins that typically comprise a soluble TCR (a polypeptide chain comprising a vα domain and a cα domain, and a polypeptide chain comprising a vβ domain and a cβ domain), wherein the vβcβ polypeptide chain is linked to a transmembrane domain and an intracellular signaling component (e.g., an ITAM-containing activation domain and optionally a costimulatory signaling domain) (see, e.g., walseng et al, 2017 science report (SCIENTIFIC REPORTS) 7:10713).

In certain embodiments, an engineered host cell that co-expresses a chimeric Tim4 receptor and a cellular immunotherapeutic (e.g., CAR, TCR, etc.) includes a recombinant nucleic acid encoding the chimeric Tim4 receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapeutic on different vectors within the engineered host cell.

In some embodiments, an engineered host cell that co-expresses a chimeric Tim4 receptor and a cellular immunotherapeutic (e.g., CAR, TCR, etc.) includes a recombinant nucleic acid encoding the chimeric Tim4 receptor and a recombinant nucleic acid molecule encoding the cellular immunotherapeutic on the same vector as the chimeric Tim4 receptor within the engineered host cell. The chimeric Tim4 receptor and the cellular immunotherapeutic agent may be expressed under the control of different promoters on the same vector (e.g., at different multiple cloning sites). Alternatively, the chimeric Tim4 receptor and cellular immunotherapeutic agent may be expressed under the control of one promoter in a polycistronic vector (e.g., a tandem expression vector). The polynucleotide sequence encoding the chimeric Tim4 receptor and the polynucleotide sequence encoding the cellular immunotherapeutic may be isolated by IRES or viral 2A peptide in a polycistronic vector.

Tandem expression cassettes, tandem expression vectors, and engineered host cells comprising them are described in international application publication No. WO2019/191339, which is incorporated herein by reference in its entirety.

In certain embodiments, the gene editing methods are used to modify the host cell genome to include a polynucleotide encoding a chimeric Tim4 receptor of the present disclosure. Gene editing or genome editing is a genetically engineered method in which DNA is inserted, replaced, or removed from the genome of a host cell using a genetically engineered endonuclease. Nucleases produce a specific double strand break at a targeted site in the genome. The endogenous DNA repair pathway of the host cell then repairs the induced break, for example, by non-homologous end joining (NHEJ) and homologous recombination. Exemplary endonucleases that can be used for gene editing include Zinc Finger Nucleases (ZFNs), transcription activator-like effector (TALE) nucleases, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas nuclease systems (e.g., CRISPR-Cas 9), homing endonucleases, or combinations thereof. Methods of disrupting or knocking out genes or gene expression in immune cells including B cells and T cells using gene editing endonucleases are known in the art and are described, for example, in international application publication No. WO 2015/066262; WO 2013/074916; WO 2014/059173; cheong et al, nat. Communication (Nat. Comm.) 2016 7:10934; chu et al, proc. Natl. Acad. Sci. USA, 2016 113:12514-12519; the methods from each of these documents are incorporated herein by reference in their entirety.

In certain embodiments, expression of an endogenous gene of the host cell is inhibited, knocked down, or knocked out. Examples of endogenous genes that can be suppressed, knocked down or knocked out in B cells include IGH, igκ, igλ, or any combination thereof. Examples of endogenous genes that can be suppressed, knocked down, or knocked out in T cells include TCR genes (TRA or TRB), HLA genes (HLA class I genes or HLA class II genes), immune checkpoint molecules (PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine 、GAL9、VISTA、CEACAM-1、CEACAM-3、CEACAM-5、PVRL2、PD-1、CTLA-4、BTLA、KIR、LAG3、TIM3、A2aR、CD244/2B4、CD160、TIGIT、LAIR-1, or PVRIG/CD 112R), or any combination thereof. Expression of the endogenous gene may be inhibited, knocked down or knocked out at the gene level, transcription level, translation level, or a combination thereof. Methods of suppressing, knocking down, or knocking out an endogenous gene can be accomplished, for example, by an RNA interfering agent (e.g., siRNA, shRNA, miRNA, etc.) or an engineered endonuclease (e.g., CRISPR/Cas nuclease system, zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), homing endonuclease (meganucleotide)), or any combination thereof. In certain embodiments, an endogenous B cell gene (e.g., IGH, igκ, or igλ) is knocked out, such as via an engineered endonuclease, by inserting a polynucleotide encoding a chimeric Tim4 receptor of the disclosure into a locus of the endogenous B cell gene. In certain embodiments, an endogenous T cell gene (e.g., a TCR gene, an HLA gene, or an immune checkpoint molecule gene) is knocked out, such as via an engineered endonuclease, by inserting a polynucleotide encoding a chimeric Tim4 receptor of the present disclosure into a locus of the endogenous T cell gene.

In certain embodiments, the host cell may be genetically modified to express a type of chimeric Tim4 receptor. In other embodiments, the host cell may express at least two or more different chimeric Tim4 receptors.

The present disclosure also provides a composition comprising a population of host cells modified with a chimeric Tim4 receptor. In certain embodiments, the population of host cells modified with the chimeric Tim4 receptor can be a population of B cells, a population of T cells, a population of natural killer cells, a population of lymphoid precursor cells, a population of antigen presenting cells, a population of dendritic cells, a population of langerhans cells, a population of bone marrow precursor cells, a population of mature bone marrow cells, or any combination thereof. Furthermore, a population of host cells of a particular cell type modified by a chimeric Tim4 receptor may be composed of one or more subtypes. For example, the population of B cells may consist of naive B cells modified with a chimeric Tim4 receptor, plasma cells, regulatory B cells, border zone B cells, follicular B cells, lymphoplasmacytoid cells, plasmablasts, memory B cells, or any combination thereof. In another example, the population of T cells may consist of CD4 ⁺ helper T cells modified with the chimeric Tim4 receptor, cd8+ effector (cytotoxic) T cells, naive (cd45ra+, CCR7+, cd62l+, cd27+, CD45 RO-) T cells, central memory (CD 45RO ⁺、CD62L⁺、CD8⁺) T cells, effector memory (cd445ra+, CD45RO-, CCR7-, CD62L-, CD 27-) T cells, T memory stem cells, regulatory T cells, mucosa-associated invariant T cells (MAIT), γδ (gd) cells, tissue resident T cells, natural killer T cells, or any combination thereof.

In certain embodiments, the population of host cells consists of cells each expressing the same chimeric Tim4 receptor. In other embodiments, the population of host cells consists of a mixture of two or more subpopulations of host cells, wherein each subpopulation expresses a different chimeric Tim4 receptor or a set of chimeric Tim4 receptors.

In certain embodiments, when preparing a host cell (e.g., a B cell or a T cell) modified with a chimeric Tim4 receptor, one or more growth factor cytokines that promote proliferation of the host cell (e.g., B cell or T cell) can be added to the cell culture. Cytokines may be human or non-human. Exemplary growth factor cytokines that may be used to promote T cell proliferation include IL-2, IL-15, and the like. Exemplary growth factor cytokines that may be used to promote B cell proliferation include CD40L, IL-2, IL-4, IL-15, IL-21, BAFF, and the like.

Prior to genetic modification of a host cell with a chimeric Tim4 receptor vector, a source of the host cell (e.g., T cell, B cell, natural killer cell, etc.) is obtained from a subject (e.g., whole blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue), and the host cell is isolated from the subject using methods known in the art. Specific host cell subsets can be collected according to known techniques and enriched or depleted by known techniques, such as affinity binding to antibodies, flow cytometry, and/or immunomagnetic selection. Following the enrichment and/or depletion step and introduction of the chimeric Tim4 receptor, in vitro expansion of the desired modified host cells can be performed according to known techniques or variants thereof that will be apparent to those skilled in the art.

The chimeric Tim4 receptors of the present disclosure confer host cell cytotoxic activity that expresses the chimeric Tim4 receptor specific for phosphatidylserine. Thus, host cells expressing chimeric Tim4 receptors are able to induce apoptosis of target cells upon binding to phosphatidylserine exposed on the surface of the target cells. In certain embodiments, host cells expressing the chimeric Tim4 receptor induce apoptosis of the target cells via: releasing granzyme, perforin, granulysin, or any combination thereof; fas ligand-Fas interaction; or both. In further embodiments, the chimeric Tim4 receptor further confers phosphatidylserine-specific phagocytic activity to a host cell expressing the chimeric Tim4 receptor. In still further embodiments, the host cell does not naturally exhibit a phagocytic phenotype prior to modification with the chimeric Tim4 receptor.

The chimeric Tim4 receptors of the disclosure are also capable of costimulating T cells via at least one signaling pathway. In certain embodiments, the chimeric Tim4 receptor provides a costimulatory signal to T cells via at least two different signaling pathways (e.g., via a costimulatory signaling domain selected in the chimeric Tim4 receptor). For example, a chimeric Tim4 receptor comprising a CD28 costimulatory signaling domain may be capable of providing a costimulatory signal via CD28 and Tim 1. In certain embodiments, host immune cells expressing the chimeric Tim4 receptor exhibit a reduction or inhibition of immune cell depletion. In certain embodiments, the host immune cell is a T cell or NK cell. In certain embodiments, the depleted T cells exhibit: (a) Increased expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof; (b) Reduced production of IFN-gamma, IL-2, TNF-alpha, or any combination thereof; or both (a) and (b). In certain embodiments, the depleted NK cells exhibit: (a) Increased expression of PD-1, NKG2A, TIM3, or any combination thereof; (b) reduced production of IFN- γ, TNF- α, or both; or both (a) and (b).

In certain embodiments, host cells expressing the chimeric Tim4 receptor exhibit an enhanced effector response (e.g., tumor-specific). In certain embodiments, the effector response is enhanced T cell proliferation, cytokine production (e.g., IFN- γ, IL-2, TNF- α), cytotoxic activity, persistence, or any combination thereof. Host cells expressing the chimeric Tim4 receptor can be administered to a subject alone or in combination with other therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiation therapies, chemotherapeutics, small molecules, oncolytic viruses, electric pulse therapies, and the like.

In certain embodiments, host cells expressing the chimeric Tim4 receptor exhibit a reduced immunosuppressive response to phosphatidylserine. Phosphatidylserine is one of the major apoptotic cell ligands that signals phagocytes of "eat me". Clearance of apoptotic cells by phagocytes generally reduces or prevents inflammatory responses via secretion of anti-inflammatory cytokines IL-10 and TGF-beta and reduction of secretion of inflammatory cytokines TNF-alpha, IL-1β and IL-12. Thus, phosphatidylserine may act as an immunosuppressive signal during the clearance of apoptotic cells. In certain embodiments, host cells modified with a chimeric Tim4 receptor exhibit increased antigen-specific cytokine production (e.g., IFN- γ, IL-2, TNF- α) upon binding to phosphatidylserine, thereby reducing an immunosuppressive response to phosphatidylserine.

In some embodiments, T cells expressing the chimeric Tim4 receptor exhibit increased or enhanced antigen capture, antigen processing, and/or antigen presentation activity. Methods for measuring the ability of chimeric Tim4 receptor T cells to present a current target peptide antigen and induce target peptide specific activation of target peptide specific T cells are described in examples 2 and 4.

Expression of the chimeric Tim4 receptor on a host cell can be functionally characterized according to any of a number of art accepted methods for determining host cell (e.g., T cell) activity, including determining T cell binding, activation or induction and also including determining antigen-specific T cell responses. Examples include determining T cell proliferation, T cell cytokine release, antigen specific T cell stimulation, CTL activity (e.g., by detecting ⁵¹ Cr or europium release from preloaded target cells), changes in T cell phenotype marker expression, and other measurements of T cell function. Methods for performing these and similar assays can be found, for example, in Lefkovits (Manual of immunological methods: technical comprehensive materials (Immunology Methods Manual: the Comprehensive Sourcebook of Techniques), 1998). See also current immunological protocols (Current Protocols in Immunology); weir, manual for laboratory immunology (Handbook of Experimental Immunology), bulleweil science publication (Blackwell Scientific), boston, mass (1986); mishell and Shigii (editions), "selected methods in cellular immunology (Selected Methods in Cellular Immunology), frieman Press (Freeman Publishing), san Francisco, california (1979); green and Reed, science 281:1309 (1998) and references cited therein. Cytokine levels can be determined according to methods known in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining, flow cytometry, and any combination thereof (e.g., a combination of intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from antigen-specific priming or stimulation of an immune response can be determined by isolating lymphocytes, such as circulating lymphocytes in a sample of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and/or cell viability, such as by incorporating tritiated thymidine or a non-radioactive assay, such as an MTT assay, and the like.

In certain embodiments, the host cell modified with the chimeric Tim4 receptor has a phagocytic index of about 20 to about 1,500 for the target cell. The "phagocytic index" is a measure of the phagocytic activity of a transduced host cell, as determined by counting the number of target cells or particles taken up by each chimeric Tim4 receptor-modified host cell during a period of time in which a suspension of target cells or particles and chimeric Tim4 receptor-modified host cells is incubated in a culture medium. The phagocytic index can be calculated by multiplying [ total number of target cells phagocytosed/total number of chimeric Tim4 receptor modified cells counted (e.g., phagocytic frequency) ]x [ average area per target cell or particle of chimeric Tim4 receptor + host cells x 100 (e.g., hybridization capture) ] or by [ total number of particles phagocytosed/total number of chimeric Tim4 receptor modified host cells counted ] × [ number of chimeric Tim4 receptor modified host cells containing particles phagocytosed/total number of chimeric Tim4 receptor cells counted ] x100. In certain embodiments, the cells modified with the chimeric Tim4 receptor have a molecular weight of about 30 to about 1,500; about 40 to about 1,500; about 50 to about 1,500; about 75 to about 1,500; about 100 to about 1,500; about 200 to about 1,500; about 300 to about 1,500; about 400 to about 1,500; about 500 to about 1,500; about 20 to about 1,400; about 30 to about 1,400; about 40 to about 1,400; about 50 to about 1,400; about 100 to about 1,400; about 200 to about 1,400; about 300 to about 1,400; about 400 to about 1,400; about 500 to about 1,400; about 20 to about 1,300; about 30 to about 1,300; about 40 to about 1,300; about 50 to about 1,300; about 100 to about 1,300; about 200 to about 1,300; about 300 to about 1,300; about 400 to about 1,300; about 500 to about 1,300; about 20 to about 1,200; about 30 to about 1,200; about 40 to about 1,200; about 50 to about 1,200; about 100 to about 1,200; about 200 to about 1,200; about 300 to about 1,200; about 400 to about 1,200; about 500 to about 1,200; about 20 to about 1,100; about 30 to about 1,100; about 40 to about 1,100; about 50 to about 1,100; about 100 to about 1,100; about 200 to about 1,100; about 300 to about 1,100; about 400 to about 1,100; or about 500 to about 1,100; about 20 to about 1,000; about 30 to about 1,000; about 40 to about 1,000; about 50 to about 1,000; about 100 to about 1,000; about 200 to about 1,000; about 300 to about 1,000; about 400 to about 1,000; or about 500 to about 1,000; about 20 to about 750; about 30 to about 750; about 40 to about 750; about 50 to about 750; about 100 to about 750; about 200 to about 750; about 300 to about 750; about 400 to about 750; or about 500 to about 750; about 20 to about 500; about 30 to about 500; about 40 to about 500; about 50 to about 500; about 100 to about 500; about 200 to about 500; or a phagocytic index of about 300 to about 500. In further embodiments, the incubation time is from about 2 hours to about 4 hours, about 2 hours, about 3 hours, or about 4 hours. In still further embodiments, the cells modified with the chimeric Tim4 receptor exhibit a phagocytic index that is statistically significantly higher than cells transduced with a truncated EGFR control. Phagocytosis index can be calculated using methods known in the art, and as further described in the examples and PCT application No. PCT/US2017/053553 (incorporated herein by reference in its entirety), including quantification by flow cytometry or fluorescence microscopy.

The host cell may be from an animal, such as a human, primate, cow, horse, sheep, dog, cat, mouse, rat, rabbit, guinea pig, or a combination thereof. In a preferred embodiment, the animal is a human. The host cell may be obtained from a healthy subject or a subject suffering from a disease associated with the expression or overexpression of an antigen.

Application method

In one aspect, the present disclosure provides a method for conferring or enhancing phosphatidylserine-specific cytotoxic activity on a cell, comprising introducing into a host cell a nucleic acid molecule encoding at least one chimeric Tim4 receptor or a chimeric Tim4 receptor vector according to any of the embodiments described herein; and expressing at least one chimeric Tim4 receptor in the host cell, wherein the at least one chimeric Tim4 receptor enhances phosphatidylserine specific cytotoxic activity of the host cell compared to the host cell prior to modification to express the chimeric Tim4 receptor. In certain embodiments, the cytotoxic activity of the host cell is increased by at least about 10％、15％、20％、25％、30％、35％、40％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％ or more as compared to the host cell prior to modification with the nucleic acid molecule encoding the chimeric Tim4 receptor or the chimeric Tim4 receptor vector. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell or NK cell. Methods for measuring the cytotoxic activity of host cells, particularly immune cells such as T cells and NK cells, include chromium (⁵¹ Cr) release assays, β -gal or firefly luciferase release assays, flow cytometry methods that mediate target cell death and effector cell activity (see, e.g., vaccine specialist reviews (Expert reviews), 2010, 9:601-616).

In certain embodiments, the method for conferring or enhancing a phosphatidylserine-specific cytotoxic activity on a cell further comprises conferring or enhancing a phosphatidylserine-specific phagocytic activity on a host cell expressing at least one chimeric Tim4 receptor. In certain such embodiments, the host cell does not naturally exhibit a phagocytic phenotype prior to modification with the chimeric Tim4 receptor. For example, in certain such embodiments, phagocytic activity of the host cell is increased by at least about 10％、15％、20％、25％、30％、35％、40％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％ or more as compared to the host cell prior to modification to express the chimeric Tim4 receptor vector. In certain embodiments, the host cell does not naturally have phagocytic activity. In some embodiments, the host cell is an immune cell. In some embodiments, the host cell is a T cell or NK cell. Methods of measuring phagocytic activity of a host cell include methods as described in international application publication No. WO2018/064076 (incorporated herein by reference in its entirety).

In another aspect, a chimeric Tim4 receptor, a polynucleotide encoding a chimeric Tim4 receptor, a chimeric Tim4 receptor vector, or a host cell expressing a chimeric Tim4 receptor according to any of the embodiments provided herein can be used in a method of enhancing effector function of a host cell. In certain embodiments, the enhanced effector function comprises enhanced cytotoxic activity, enhanced antigen-specific cytokine production (e.g., IFN- γ, IL-2, TNF- α, or any combination thereof), enhanced anti-apoptotic signaling, enhanced persistence, enhanced amplification, enhanced proliferation, or any combination thereof. In certain embodiments, the effector function of the host cell is enhanced by at least about 10％、15％、20％、25％、30％、35％、40％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％ or more as compared to a host cell not modified with a nucleic acid molecule encoding the chimeric Tim4 receptor or a chimeric Tim4 receptor vector. In some embodiments, the host cell is an immune cell. In certain embodiments, the host cell is a T cell or NK cell.

In another aspect, host cells modified with the chimeric Tim4 receptors of the present disclosure can be used in methods of inhibiting or reducing immune cell depletion. In some embodiments, the immune cell is a T cell or NK cell. In certain embodiments, the reduced depletion in T cells comprises: (a) Reduced expression of PD-1, TIGIT, LAG3, TIM3, or any combination thereof in T cells; (b) Increased production of IFN-gamma, IL-2, TNF-alpha, or any combination thereof in T cells; or both (a) and (b). In certain embodiments, the reduced depletion in NK cells comprises: (a) Reduced expression of PD-1, NKG2A, TIM, or any combination thereof in NK cells; (b) Increased production of IFN-gamma, TNF-alpha, or both in NK cells; or both (a) and (b). In certain embodiments, expression of the immune checkpoint molecule is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in a host immune cell expressing the chimeric Tim4 receptor as compared to a host immune cell not modified with a nucleic acid molecule encoding the chimeric Tim4 receptor or a chimeric Tim4 receptor vector. In certain embodiments, the expression of the cytokine is increased by at least about 5％、10％、15％、20％、25％、30％、35％、40％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％、110％、120％、130％、140％、150％、160％、170％、180％、190％、200％ or more in a host immune cell expressing the chimeric Tim4 receptor as compared to a host immune cell not modified with a nucleic acid molecule encoding the chimeric Tim4 receptor or a chimeric Tim4 receptor vector.

In another aspect, a chimeric Tim4 receptor, a polynucleotide encoding a chimeric Tim4 receptor, a chimeric Tim4 receptor vector, or a host cell expressing a chimeric Tim4 receptor according to any of the embodiments provided herein can be used in a method of reducing an immunosuppressive response to phosphatidylserine in a host cell. In certain embodiments, the immunosuppressive response includes secretion of anti-inflammatory cytokines (e.g., IL-10, TGF-beta, or both), reduction of secretion of inflammatory cytokines (e.g., TNF-alpha, IL-1 beta, and IL-12), or both. In certain embodiments, the immunosuppressive response of a host cell to phosphatidylserine is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% as compared to a host cell not modified with a nucleic acid molecule encoding a chimeric Tim4 receptor or a chimeric Tim4 receptor vector. In some embodiments, the host cell is an immune cell. In certain embodiments, the host cell is a T cell or NK cell.

In yet other aspects, the chimeric Tim4 receptor, polynucleotide encoding the chimeric Tim4 receptor, chimeric Tim4 receptor vector, or host cell expressing the chimeric Tim4 receptor according to any of the embodiments provided herein can be used in a method of eliminating target cells with surface-exposed phosphatidylserine, e.g., for eliminating cancer cells with surface-presented phosphatidylserine. In certain embodiments, the target cell is a damaged, stressed, apoptotic, necrotic cell (e.g., a tumor cell) with surface exposed phosphatidylserine. In certain embodiments, host cells expressing the chimeric Tim4 receptor clear damaged, stressed, apoptotic, or necrotic target cells with surface exposed phosphatidylserine via induction of apoptosis or induction of both apoptosis and phagocytosis. Host cells expressing the chimeric Tim4 receptor can be administered to a subject alone or in combination with other therapeutic agents including, for example, CAR-T cells, TCRs, antibodies, radiation therapies, chemotherapy, small molecules, oncolytic viruses, electric pulse therapies, and the like.

In another aspect, the chimeric Tim4 receptor, polynucleotide encoding the chimeric Tim4 receptor, chimeric Tim4 receptor vector, or host cell expressing the chimeric Tim4 receptor according to any of the embodiments provided herein can be used in a method of enhancing the effect of a therapeutic agent that induces stress, injury, necrosis, or apoptosis in a cell. Certain therapies, such as chemotherapy, radiation therapy, UV light therapy, electrical pulse therapy, adoptive cell immunotherapy (e.g., CAR-T cells, TCRs) and oncolytic virus therapy, can induce cell damage or death of tumor cells, diseased cells and cells in their surroundings. Cells expressing the chimeric Tim4 receptor can be administered in combination with cell injury/cytotoxicity therapy to bind to phosphatidylserine moieties exposed on the outer leaflet of the targeted cells and to clear stressed, injured, diseased, apoptotic, necrotic cells.

In another aspect, the present disclosure provides a method for conferring or enhancing antigen capture, antigen processing and/or antigen presentation activity of a cell, comprising introducing into a host cell a nucleic acid molecule encoding at least one chimeric Tim4 receptor or a chimeric Tim4 receptor vector according to any of the embodiments described herein; and expressing at least one chimeric Tim4 receptor in the host cell, wherein the at least one chimeric Tim4 receptor enhances antigen capture, antigen processing, and/or antigen presentation activity of the host cell as compared to the host cell prior to modification to express the chimeric Tim4 receptor.

The chimeric Tim4 receptor, polynucleotide encoding the chimeric Tim receptor, chimeric Tim receptor vector, host cell expressing the chimeric Tim receptor, or pharmaceutical composition thereof according to any of the embodiments provided herein, can be used in a method of enhancing ccr7+ expressing T cells in a subject having cancer, optionally in combination with a PARP inhibitor. In some embodiments, the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer, or melanoma. In some embodiments, the chimeric Tim4 receptor is a single-chain chimeric protein comprising: (a) an extracellular domain comprising a Tim4 binding domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, a CD3 zeta signaling domain, and a TLR2 TIR signaling domain; and (c) a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

The enhancement of CCR7 expression on T cells expressing chimeric Tim4 can be at least 10％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％、125％、150％、175％、200％、225％、250％、275％、300％、325％、350％、375％、400％ or more CCR7 expression compared to expression on T cells expressing chimeric Tim4 without PARP inhibitor administration.

The chimeric Tim4 receptor, polynucleotide encoding the chimeric Tim receptor, chimeric Tim receptor vector, host cell expressing the chimeric Tim receptor, or pharmaceutical composition thereof according to any of the embodiments provided herein, can be used in a method of enhancing CD4/CD 8T cell ratio in a subject having cancer, optionally in combination with a PARP inhibitor. In some embodiments, the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer, or melanoma. In some embodiments, the chimeric Tim4 receptor is a single-chain chimeric protein comprising: (a) an extracellular domain comprising a Tim4 binding domain; (b) An intracellular signaling domain comprising a CD28 signaling domain, a CD3 zeta signaling domain, and a TLR2TIR signaling domain; and (c) a CD28 transmembrane domain located between and connecting the extracellular domain and the intracellular signaling domain.

The enhanced CD4/CD8T cell ratio in T cells expressing chimeric Tim4 can be at least 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or higher CD4/CD8T cell ratio compared to expression on T cells expressing chimeric Tim4 without PARP inhibitor administration.

The chimeric Tim receptor, polynucleotide encoding the chimeric Tim receptor, chimeric Tim receptor vector, or host cell expressing the chimeric Tim receptor according to any of the embodiments provided herein, in combination with a PARP inhibitor, can be used in a method of treating a subject having a disease, disorder, or undesired condition. Embodiments of these methods comprise administering to a subject (i) a therapeutically effective amount of a pharmaceutical composition comprising one or more chimeric Tim receptors, a polynucleotide encoding one or more chimeric Tim receptors, a vector comprising a polynucleotide encoding one or more chimeric Tim receptors, or a population of host cells genetically modified to express one or more chimeric Tim receptors according to the present description; and (ii) a therapeutically effective amount of a pharmaceutical composition comprising a PARP inhibitor.

The chimeric Tim4 receptor compositions as described herein can be administered prior to (e.g., 1 day to 30 days or more prior to) PARP inhibitor therapy, concurrently with (on the same day) PARP inhibitor therapy, or after (e.g., 1 day to 30 days or more after) PARP inhibitor therapy. In certain embodiments, the cells modified with the chimeric Tim4 receptor are administered after the PARP inhibitor is administered. In further embodiments, the cells modified with the chimeric Tim4 receptor are administered 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, or 30 days after administration of the PARP inhibitor. In still further embodiments, the cells modified with the chimeric Tim4 receptor are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the PARP inhibitor therapy. Where PARP inhibitor therapy involves multiple doses, the chimeric Tim receptor modified cells may be administered after an initial dose of PARP inhibitor, after a final dose of PARP inhibitor, or between doses of PARP inhibitor.

In another aspect, a chimeric Tim4 receptor, a polynucleotide encoding a chimeric Tim4 receptor, a chimeric Tim4 receptor vector, or a host cell expressing a chimeric Tim4 receptor according to any of the embodiments provided herein can be used in a method of treating a subject having a disease, disorder, or undesired condition. Embodiments of these methods comprise administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising one or more chimeric Tim4 receptors, a polynucleotide encoding one or more chimeric Tim4 receptors, a vector comprising a polynucleotide encoding one or more chimeric Tim4 receptors, or a population of host cells genetically modified to express one or more chimeric Tim4 receptors according to the present description.

Diseases that can be treated with cells expressing the chimeric Tim4 receptor as described in the present disclosure include cancer and infectious diseases (viral, bacterial, fungal, protozoal infections). Adoptive immunity and gene therapy are promising treatments for various types of cancer (Morgan et al, science 314:126, 2006; schmitt et al, human gene therapy 20:1240, 2009; june, journal of clinical research (J.Clin. Invest.) 117:1466, 2007) and infectious diseases (Kitchen et al, public science library. Complex. 4:38208, 2009; rossi et al, natural biotechnology 25:1444, 2007; zhang et al, PLoS Pathos.) > 6:e1001018, 2010; luo et al, journal of molecular medicine (J.mol. Med.)) 89:903, 2011).

A variety of cancers (including solid tumors and leukemia) are suitable for use in the compositions and methods disclosed herein. Exemplary cancers that can be treated using the receptors, modified host cells, and compositions described herein include adenocarcinomas of the breast, prostate, and colon; all forms of lung bronchogenic carcinoma; myeloid leukemia; melanoma; liver cancer; neuroblastoma; papillomas; amine precursor uptake and decarboxylation of the cell tumor; a vaginosis tumor; gill tumor; malignant carcinoid syndrome; carcinoid heart disease; and cancers (e.g., walker cancer, basal cell carcinoma, basal squamous cell carcinoma, brown-Pearce cancer, ductal carcinoma, ehrlich tumor, krebs2 cancer, merkel cell carcinoma, mucinous cancer, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, hard carcinoma, bronchiolar carcinoma, squamous cell carcinoma, and transitional cell carcinoma). Other cancer types that can be treated using the receptors, modified host cells, and compositions described herein include histiocyte disorders; malignant tissue cytopathy; leukemia; hodgkin's disease; immunoproliferative small bowel disease; non-hodgkin's lymphoma; plasmacytoma; multiple myeloma; chronic Myelogenous Leukemia (CML); acute Myelogenous Leukemia (AML); plasmacytoma; reticuloendothelial tissue proliferation; melanoma; chondroblastoma; cartilage tumor; chondrosarcoma; fibroids; fibrosarcoma; giant cell tumor; histiocytoma; a fatty tumor; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngeal pipe tumor; a vegetative cell tumor; hamartoma; a stromal tumor; mesonephroma; myosarcoma; enameloblastoma; cementoma; dental tumor; teratoma; thymoma; nourishing cell tumor. Furthermore, the following types of cancers are also contemplated as suitable for treatment using the receptors, modified host cells, and compositions described herein: adenoma; gall bladder tumor; cholesteatoma; cylindrical tumors; cystic adenocarcinoma; cystic adenoma; granulosa cell tumors; ampholytic embryonal cytoma; liver cancer; sweat gland tumor; islet cell tumor; leydig cell tumor; papillomas; support cell tumor; follicular membrane cytoma; smooth myoma; leiomyosarcoma; myoblasts; myomas; myosarcoma; rhabdomyomas; rhabdomyosarcoma; ventricular tube membranoma; gangliocytoma; glioma; medulloblastoma; meningioma; a schwannoma; neuroblastoma; neuroepithelial tumors; neurofibromatosis; neuroma; paraganglioma; non-chromaphilic paragangliomas. The types of cancers that can be treated also include angiokeratomas; vascular lymphoid hyperplasia is accompanied by eosinophilia; sclerosing hemangioma; hemangiomatosis; glomeroclavicular tumor; vascular endothelial tumors; hemangioma; vascular endothelial cell tumor; hemangiosarcoma; lymphangioma; lymphangiomyomas; lymphangiosarcoma; pineal tumor; carcinoma sarcoma; chondrosarcoma; she Zhuangnang sarcoma; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukemia sarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasms; neurofibromatosis; cervical dysplasia and peritoneal cancer.

Examples of hyperproliferative disorders suitable for therapy using the receptors, modified host cells, and compositions described herein include B cell cancers (B cell malignancies), including B-cell lymphomas (such as various forms of hodgkin's disease, non-hodgkin's lymphoma (NHL) or central nervous system lymphoma), leukemias (such as Acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, B-cell transformation of chronic myelogenous leukemia, acute Myelogenous Leukemia (AML), chronic myelogenous leukemia, and myelomas (such as multiple myeloma). Additional B-cell cancers that can be treated using the receptors, modified host cells, and compositions described herein include small lymphocytic lymphomas, small lymphocytic leukemia, megaloblastic, B-cell pre-lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphomas, plasmacytic myelomas, bone solitary plasmacytic myelomas, extraosseous plasmacytic cell lymphomas, perinodal marginal zone B-cell lymphomas of mucosa-associated (MALT) lymphoid tissue, junction marginal zone B-cell lymphomas, follicular lymphomas, mantle cell lymphomas, diffuse large B-cell lymphomas, thymic (thymic) large B-cell lymphomas, B-cell mass, B-cell lymphomas, primary lymphomas, lymphomas of primary lymphomas, granulomatosis, and granulomatosis, lymphomas of the proliferation, and granulomatosis.

In some embodiments, combination therapies comprising a chimeric Tim4 receptor, a polynucleotide encoding a chimeric Tim4 receptor, a chimeric Tim4 receptor vector, or a host cell expressing a chimeric Tim4 receptor according to any of the embodiments provided herein, and a PARP inhibitor can be used to treat a solid tumor. In some embodiments, the solid tumor cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, or prostate cancer. In some embodiments, the breast cancer is a triple negative breast cancer. In some embodiments, the ovarian cancer is advanced ovarian cancer. In some embodiments, the prostate cancer is advanced prostate cancer. In some embodiments, the solid tumor cancer is melanoma. In some embodiments, the solid tumor cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the solid tumor cancer is a breast cancer (BRCA) mutant cancer. In some embodiments, the cancer is BRCA1 mutant cancer, BRCA2 mutant cancer, or both.

Infectious diseases include those associated with infectious agents and include any of a variety of bacteria (e.g., pathogenic escherichia coli, salmonella typhimurium (s. Tyrphium), pseudomonas aeruginosa (p. Aerospora), bacillus anthracis (b. Anthracis), clostridium botulinum (c. Botulium), clostridium difficile (c. Diffiie), clostridium perfringens (c. Perfringens), helicobacter pylori (h. Pyrri), vibrio cholerae (v. Cholerae), listeria species (Listeria spp.), rickettsia species (Rickettsia spp.), chlamydia species (Chlamydia spp.), and the like), mycobacteria, and parasites (including any known parasite members of protozoa). Infectious viruses include eukaryotic viruses such as adenovirus, bunyavirus, herpesvirus, papovavirus, papillomavirus (e.g., HPV), paramyxovirus, picornavirus, rhabdovirus (e.g., rabies virus), orthomyxovirus (e.g., influenza virus), poxvirus (e.g., vaccinia virus), reovirus, retrovirus, lentivirus (e.g., HIV), flavivirus (e.g., HCV, HBV), and the like. In certain embodiments, compositions comprising chimeric Tim4 receptors according to the present disclosure are used to treat infections of microorganisms capable of establishing persistent infections in a subject.

The chimeric Tim4 receptors of the disclosure can be administered to a subject in a cell-bound form (e.g., gene therapy of a target cell population). Thus, for example, the chimeric Tim4 receptors of the present disclosure can be administered to a subject expressed on the surface of T cells, natural killer T cells, B cells, lymphoid precursor cells, antigen presenting cells, dendritic cells, langerhans cells, bone marrow precursor cells, mature bone marrow cells (including subpopulations thereof), or any combination thereof. In certain embodiments, the method of treating a subject comprises administering an effective amount of cells modified with a chimeric Tim4 receptor (i.e., recombinant cells expressing one or more chimeric Tim4 receptors). The cells modified with the chimeric Tim4 receptor can be allogeneic, syngeneic, allogeneic or autologous to the subject.

Pharmaceutical compositions comprising cells modified with chimeric Tim4 receptors may be administered in a manner appropriate for the disease or condition to be treated (or prevented), as determined by one of skill in the medical arts. The appropriate dosage, appropriate duration and frequency of administration of the composition will be determined by factors such as the condition, body type, body weight, body surface area, age, sex, type and severity of the disease of the patient, the particular therapy to be administered, the particular form of the active ingredient, the time and method of administration, and other drugs being administered simultaneously. The present disclosure provides pharmaceutical compositions comprising cells modified with a chimeric Tim4 receptor and a pharmaceutically acceptable carrier, diluent or excipient. Suitable excipients include water, saline, dextrose, glycerol, and the like, as well as combinations thereof. Other suitable infusion media may be any isotonic media formulation, including saline, normosol R (Abbott), plasma-Lyte A (Baxter), 5% dextrose in water, or lactated ringer's solution.

A therapeutically effective amount of a cell in the pharmaceutical composition is at least one cell (e.g., a T cell modified with a chimeric Tim4 receptor) or more typically greater than 10 ² cells, e.g., up to 10 ⁶, up to 10 ⁷, up to 10 ⁸ cells, up to 10 ⁹ cells, up to 10 ¹⁰ cells, or up to 10 ¹¹ cells or more. In certain embodiments, the cells are administered in the range of about 10 ⁶ to about 10 ¹⁰ cells/m ², preferably in the range of about 10 ⁷ to about 10 ⁹ cells/m ². The number of cells will depend on the end use for which the composition is intended and the type of cells contained therein. For example, a composition comprising cells modified to contain a chimeric Tim4 receptor will comprise a population of cells comprising about 5% to about 95% or more of such cells. In certain embodiments, compositions comprising cells modified with a chimeric Tim4 receptor include cell populations comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the uses provided herein, the volume of the cells is typically 1 liter or less, 500ml or less, 250ml or less, or 100ml or less. Thus, the desired cell density is typically greater than 10 ⁴ cells/ml and typically greater than 10 ⁷ cells/ml, typically greater than 10 ⁸ cells/ml or greater. The cells may be administered as a single infusion or as multiple infusions over a period of time. Repeated infusions of cells modified with chimeric Tim4 receptors may be spaced days, weeks, months or even years apart if there is a recurrence of the disease or disease event. A clinically relevant number of immune cells may be distributed into multiple infusions that accumulate equal to or exceeding 10 ⁶、10⁷、10⁸、10⁹、10¹⁰ or 10 ¹¹ cells. Preferred doses for administration of host cells comprising the recombinant expression vectors as described herein are about 10 ⁷ cells/m ², about 5×10 ⁷ cells/m ², about 10 ⁸ cells/m ², about 5×10 ⁸ cells/m ², about 10 ⁹ cells/m ², about 5×10 ⁹ cells/m ², about 10 ¹⁰ cells/m ², about 5×10 ¹⁰ cells/m ² or about 10 ¹¹ cells/m ².

The chimeric Tim4 receptor compositions as described herein can be administered intravenously, intraperitoneally, intranasally, intratumorally into bone marrow, lymph nodes, and/or cerebrospinal fluid.

The chimeric Tim4 receptor composition can be administered to a subject in combination with one or more additional therapeutic agents. Examples of therapeutic agents that may be administered in combination with a chimeric Tim composition according to the present description include radiation therapies, adoptive cellular immunotherapeutic agents (e.g., recombinant TCRs, affinity-enhanced TCR, CAR, TCR-CARs, scTCR fusion proteins, dendritic cell vaccines), antibody therapies, immune checkpoint molecule inhibitor therapies, UV light therapies, electrical pulse therapies, high intensity focused ultrasound therapies, oncolytic virus therapies, or drug therapies, such as chemotherapeutic agents, therapeutic peptides, hormones, aptamers, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, small molecule therapies, or any combination thereof. In certain embodiments, host cells modified with chimeric Tim4 receptors can clear stressed, damaged, apoptotic, necrotic, infected, dead cells exhibiting surface phosphatidylserine that are induced by one or more additional therapeutic agents.

In certain embodiments, the chimeric Tim4 receptor and the adoptive cellular immunotherapeutic (e.g., CAR, TCR-CAR, TCR, etc., as described above) are administered to a subject in the same host cell or in different host cells. In certain embodiments, the chimeric Tim4 receptor and the adoptive cellular immunotherapeutic are expressed in the same host cell from the same vector or from different vectors. In certain embodiments, the chimeric Tim4 receptor and the adoptive cellular immunotherapeutic are expressed in the same host cell from a polycistronic vector. In certain embodiments, the chimeric Tim4 receptor is expressed in the same host cell type as the adoptive cellular immunotherapeutic (e.g., the chimeric Tim4 receptor is expressed in CD 4T cells and the CAR/or TCR is expressed in CD 4T cells, or the chimeric Tim4 receptor is expressed in CD 8T cells and the CAR/or TCR is expressed in CD 8T cells). In other embodiments, the chimeric Tim4 receptor is expressed in a different host cell type as an adoptive immunotherapeutic (e.g., the chimeric Tim4 receptor is expressed in CD 4T cells and the CAR/or TCR is expressed in CD 8T cells). Cellular immunotherapy compositions, methods of manufacture, and methods of use comprising combinations of immune cells or cell subsets engineered with chimeric Tim4 receptors and cellular immunotherapeutic agents (e.g., CARs, TCRs, etc.) are described in PCT international publication No. WO2019/191340, which is incorporated herein by reference in its entirety.

Exemplary antigens that recombinant TCR, affinity enhanced TCR, CAR, TCR-CAR or scTCR fusion proteins can target include WT-1, mesothelin, MART-1, NY-ESO-1, MAGE-A3, HPV E7, survivin, alpha fetoprotein and tumor specific neoantigens.

The CARs of the present disclosure can target a variety of antigens, including viral antigens, bacterial antigens, fungal antigens, parasitic antigens, tumor antigens, autoimmune disease antigens. Exemplary antigens that a CAR may target include CD138, CD38, CD33, CD123, CD72, CD79a, CD79B, mesothelin 、PSMA、BCMA、ROR1、MUC-16、L1CAM、CD22、CD19、CD20、CD23、CD24、CD37、CD30、CA125、CD56、c-Met、EGFR、GD-3、HPV E6、HPV E7、MUC-1、HER2、 folate receptor alpha, CD97, CD171, CD179a, CD44v6, WT1, VEGF-alpha, VEGFR1, IL-13 ra 2, IL-11 ra, PSA, fcRH5, NKG2D ligand, NY-ESO-1, TAG-72, CEA, ephrin A2, ephrin B2, lewis a antigen, lewis Y antigen, MAGE-A1, RAGE-1, folate receptor beta, EGFRviii, VEGFR-2, LGR5, SSX2, AKAP-4, FLT3, fucosyl GM1, GM3, o-acetyl-GD 2, and GD2.

In some embodiments, the chimeric Tim4 receptor of the disclosure is administered to a subject in combination with a CD 72-specific CAR. In some embodiments, the binding domain of the CD72 specific CAR comprises:

(i) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 101; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 102; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 103; and a light chain Variable (VL) region, wherein the VL region comprises a light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 104; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 105; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 106;

(ii) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 107; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 108; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 109; and a light chain Variable (VL) region, wherein the VL region comprises a light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 110; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 111; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 112;

(iii) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID NO: 302; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 303; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 304; and a light chain Variable (VL) region, wherein the VL region comprises light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 305; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 306; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 307;

(iv) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 308; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence shown in SEQ ID NO. 309; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 310; and a light chain Variable (VL) region, wherein the VL region comprises light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 311; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 312; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 313;

(v) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 314; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 315; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 316; and a light chain Variable (VL) region, wherein the VL region comprises light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 317; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence shown in SEQ ID NO. 318; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence shown in SEQ ID NO: 319;

(vi) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID NO: 320; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 321; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 322; and a light chain Variable (VL) region, wherein the VL region comprises light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 323; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 324; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 325;

(vii) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 326; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO 327; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 328; and a light chain Variable (VL) region, wherein the VL region comprises light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 329; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 330; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 331;

(viii) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 332; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence shown in SEQ ID NO. 333; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 334; and a light chain Variable (VL) region, wherein the VL region comprises a light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 335; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence shown in SEQ ID NO: 336; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 337;

(ix) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 338; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO 339; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 340; and a light chain Variable (VL) region, wherein the VL region comprises a light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 341; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 342; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 343; or alternatively

(X) A heavy chain Variable (VH) region, wherein the VH region comprises heavy chain complementarity determining region 1 (HCDR-1) comprising the amino acid sequence shown in SEQ ID No. 344; heavy chain complementarity determining region 2 (HCDR-2) comprising the amino acid sequence set forth in SEQ ID NO. 345; and heavy chain complementarity determining region 3 (HCDR-3) comprising the amino acid sequence set forth in SEQ ID NO. 346; and a light chain Variable (VL) region, wherein the VL region comprises light chain complementarity determining region 1 (LCDR-1) comprising the amino acid sequence set forth in SEQ ID NO. 347; light chain complementarity determining region 2 (LCDR-2) comprising the amino acid sequence shown in SEQ ID NO. 348; and light chain complementarity determining region 3 (LCDR-3) comprising the amino acid sequence shown in SEQ ID NO. 349.

In some embodiments, the binding domain of the CAR comprises:

(i) A VH region comprising the amino acid sequence shown in SEQ ID No. 113 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 113, and a VL region comprising the amino acid sequence shown in SEQ ID No. 114 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 114;

(ii) A VH region comprising the amino acid sequence shown in SEQ ID No. 115 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 115, and a VL region comprising the amino acid sequence shown in SEQ ID No. 116 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 116;

(iii) A VH region comprising the amino acid sequence shown in SEQ ID No. 117 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 117, and a VL region comprising the amino acid sequence shown in SEQ ID No. 118 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 118;

(iv) A VH region comprising the amino acid sequence shown in SEQ ID No. 119 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 119, and a VL region comprising the amino acid sequence shown in SEQ ID No. 120 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 120;

(v) A VH region comprising the amino acid sequence shown in SEQ ID No. 121 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 121, and a VL region comprising the amino acid sequence shown in SEQ ID No. 122 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 122;

(vi) A VH region comprising the amino acid sequence shown in SEQ ID No. 123 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 123, and a VL region comprising the amino acid sequence shown in SEQ ID No. 124 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 124;

(vii) A VH region comprising the amino acid sequence shown in SEQ ID No. 125 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 125, and a VL region comprising the amino acid sequence shown in SEQ ID No. 126 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 126;

(viii) A VH region comprising the amino acid sequence set forth in SEQ ID No. 127 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 127, and a VL region comprising the amino acid sequence set forth in SEQ ID No. 128 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 128; or alternatively

(Ix) A VH region comprising the amino acid sequence shown in SEQ ID No. 129 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 129, and a VL region comprising the amino acid sequence shown in SEQ ID No. 130 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID No. 130.

In some embodiments, the VH region and the VL region are connected by a flexible linker. In some embodiments, the binding domain comprises an scFv comprising a VH region, a VL region, and a flexible linker, which may be in a VH-linker-VL orientation or a VL-linker-VH orientation. In some embodiments, the flexible linker has a length of about 5 to about 50 amino acids and includes a glycine, serine, and/or threonine rich sequence. Exemplary linkers include linkers having (GGGGS) _x or (GGGS) _x, where x = 2-5. In some embodiments, the flexible linker comprises the amino acid sequence set forth in any one of SEQ ID NOS 165-170.

In some embodiments, the binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOS.131-164 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS.131-164.

In certain embodiments, the extracellular domain of a CAR provided in the present disclosure optionally includes an extracellular non-signaling spacer or linker domain between the binding domain and the transmembrane domain. Such spacers or linker domains, if included, can position the binding domain away from the host cell surface to further achieve proper cell-to-cell contact, binding, and activation. The extracellular spacer domain is typically located between the extracellular binding domain and the transmembrane domain of the CAR. The length of the extracellular spacer may be varied to optimize target molecule binding based on the selected target molecule, the selected binding epitope, the binding domain size and affinity (see, e.g., guest et al, journal of immunotherapy, 28:203-11, 2005; pct publication No. WO 2014/031687). In certain embodiments, the extracellular spacer domain is an immunoglobulin hinge region (e.g., igG1, igG2, igG3, igG4, igA, igD). The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. An altered IgG ₄ hinge region is described in PCT publication No. WO 2014/031687, the hinge region of which is incorporated herein by reference in its entirety. In some embodiments, the extracellular spacer domain comprises a modified IgG ₄ hinge region having an amino acid sequence of ESKYGPPCPPCP (SEQ ID NO: 9).

Other examples of hinge regions that can be used in the CARs described herein include hinge regions from extracellular regions of type 1 membrane proteins (e.g., CD8a, CD4, CD28, and CD7, which can be wild-type or variants thereof). An exemplary CD8a hinge region includes the amino acid sequence shown in SEQ ID NO. 22. An exemplary CD28 hinge region includes the amino acid sequence shown in SEQ ID NO. 10. In some embodiments, the extracellular spacer domain comprises all or a portion of an immunoglobulin Fc domain selected from a CH1 domain, a CH2 domain, a CH3 domain, or a combination thereof (see, e.g., PCT publication WO2014/031687, the spacers of which are incorporated herein by reference in their entirety). In yet further embodiments, the extracellular spacer domain may include a handle region of a type II C-lectin (an extracellular domain located between the C-type lectin domain and the transmembrane domain). Type II C-lectins comprise CD23, CD69, CD72, CD94, NKG2A and NKG2D.

The CARs of the disclosure include a transmembrane domain connecting and between an extracellular domain and an intracellular signaling domain. The transmembrane domain ranges from about 15 amino acids to about 30 amino acids in length. The transmembrane domain is a hydrophobic alpha helix that passes through the host cell membrane and anchors the CAR in the host cell membrane. The transmembrane domain may be fused directly to the binding domain or extracellular spacer domain, if present. In certain embodiments, the transmembrane domain is derived from an intact membrane protein (e.g., receptor, cluster of Differentiation (CD) molecule, enzyme, transporter, cell adhesion molecule, etc.). The transmembrane domain may be selected from the same molecule as the extracellular domain or intracellular signaling domain (e.g., a CAR comprising a CD28 costimulatory signaling domain and a CD28 transmembrane domain). In some embodiments, the transmembrane domain and extracellular domain are each selected from different molecules. In some embodiments, the transmembrane domain and intracellular signaling domain are each selected from different molecules. In yet other embodiments, the transmembrane domain, extracellular domain and intracellular signaling domain are each selected from different molecules.

Exemplary transmembrane domains for CARs of the present disclosure include CD28、CD2、CD4、CD8a、CD5、CD3ε、CD3δ、CD3ζ、CD9、CD16、CD22、CD25、CD27、CD33、CD37、CD40、CD45、CD64、CD79A、CD79B、CD80、CD86、CD95(Fas)、CD134(OX40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD154(CD40L)、CD200R、CD223(LAG3)、CD270(HVEM)、CD272(BTLA)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、CD279(PD-1)、CD300、CD357(GITR)、A2aR、DAP10、FcRα、FcRβ、FcRγ、Fyn、GAL9、KIR、Lck、LAT、LRP、NKG2D、NOTCH1、NOTCH2、NOTCH3、NOTCH4、PTCH2、ROR2、Ryk、Slp76、SIRPα、pTα、TCRα、TCRβ、TIM3、TRIM、LPA5 and Zap70 transmembrane domains. An exemplary CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 174. Exemplary CD28 transmembrane domains include the amino acid sequence of SEQ ID NO. 11.

The intracellular signaling domain of the CAR is an intracellular effector domain and is capable of transmitting a functional signal to a cell in response to binding of the extracellular domain of the CAR to a target molecule (e.g., CD 72) and activating at least one of a normal effector function or response of an immune cell (e.g., a T cell engineered to express the CAR). In some embodiments, the CAR induces a function of the T cell, such as cytolytic activity or T helper cell activity, such as secretion of cytokines or other factors. An intracellular signaling domain may be any portion of an intracellular signaling molecule that retains sufficient signaling activity. In some embodiments, the intracellular signaling domain is obtained from an antigen receptor component (e.g., TCR) or a co-stimulatory molecule. In some embodiments, the full-length intracellular signaling domain of an antigen receptor or co-stimulatory molecule is used. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor or co-stimulatory molecule is used, provided that the truncated portion retains sufficient signaling activity. In further embodiments, the intracellular signaling domain is a variant of the full length or truncated portion of the intracellular signaling domain of the antigen receptor co-stimulatory molecule, provided that the variant retains sufficient signaling activity (i.e., is a functional variant).

In certain embodiments, the intracellular signaling domain of the CAR comprises a signaling domain comprising an immune receptor tyrosine activation motif (ITAM). The signaling domain containing ITAM typically contains at least one (one, two, three, four or more) ITAM, which refers to a conserved motif of YXXL/I-X _6-8 -YXXL/I. The signaling domain containing ITAM can initiate T cell activation signaling following antigen binding or ligand binding. The ITAM signaling domain comprises intracellular signaling domains such as cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD278 (ICOS), DAP10, DAP12, fcrγ, and CD66 d. Exemplary CD3 zeta signaling domains that can be used in the CARs of the present disclosure include the amino acid sequences of SEQ ID NO:177 or 178.

The CAR intracellular signaling domain optionally includes a costimulatory signaling domain that, when activated with a primary or classical (e.g., ITAM-driven) activation signal, promotes or enhances a T cell response, such as T cell activation, cytokine production, proliferation, differentiation, survival, effector function, or a combination thereof. The co-stimulatory signaling domain for the CAR comprises, for example CD27、CD28、CD40L、GITR、NKG2C、CARD1、CD2、CD7、CD27、CD30、CD40、CD54(ICAM)、CD83、CD134(OX-40)、CD137(4-1BB)、CD150(SLAMF1)、CD152(CTLA4)、CD223(LAG3)、CD226、CD270(HVEM)、CD273(PD-L2)、CD274(PD-L1)、CD278(ICOS)、DAP10、LAT、LFA-1、LIGHT、NKG2C、SLP76、TRIM、ZAP70 or any combination thereof. In particular embodiments, the costimulatory signaling domain comprises an OX40, CD2, CD27, CD28, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), or 4-1BB (CD 137) signaling domain. Exemplary CD28 co-stimulatory signaling domains that may be used in the CARs of the present disclosure include the amino acid sequence shown in SEQ ID NO:179 or SEQ ID NO:180 (variants containing the L186G, L187G mutation of the native CD28 protein). An exemplary 4-1BB costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 181. In certain embodiments, the CAR comprises one, two, or more co-stimulatory signaling domains.

In some embodiments, the CAR of the present disclosure is a first generation CAR, a second generation CAR, or a third generation CAR. First generation CARs typically have an intracellular signaling domain comprising cd3ζ, fcγri, or other intracellular signaling domain containing an activation domain of ITAM to provide a T cell activation signal. The second generation CAR further comprises a costimulatory signaling domain (e.g., from an endogenous T cell costimulatory receptor, such as CD28, 4-1BB, or ICOS). The third generation CAR includes an activation domain comprising ITAM, a first costimulatory signaling domain, and a second costimulatory signaling domain.

The CARs of the present disclosure can include polynucleotide sequences from any mammalian species, including humans, primates, cows, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, transgenic species thereof, or any combination thereof. In some embodiments, the chimeric antigen receptor is murine, chimeric, human or humanized.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 131 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 182 or SEQ ID NO. 182 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 183 or SEQ ID NO. 183 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 131 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:184 or SEQ ID NO:184 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:185 or SEQ ID NO:185 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 131 and an extracellular spacer domain comprising a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 186 or SEQ ID NO. 186 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising a CD28 hinge, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 187 or SEQ ID NO. 187 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 131 and an extracellular spacer domain comprising a CD28 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:188 or SEQ ID NO:188 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising a CD28 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 189 or SEQ ID NO. 189 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 131 and an extracellular spacer domain comprising an IgG4 hinge, a CD8a transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 190 or SEQ ID NO. 190 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD8a transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 191 or SEQ ID NO. 191 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 131 and an extracellular spacer domain comprising a CD8a hinge, a CD8a transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:192 or SEQ ID NO:192 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising a CD8a hinge, a CD8a transmembrane domain, a CD28 costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:193 or SEQ ID NO:193 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence shown in SEQ ID No. 131 and an extracellular spacer domain comprising an IgG4 hinge, a CD8a transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:194 or SEQ ID NO:194 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD8a transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:195 or SEQ ID NO:195 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 131 and an extracellular spacer domain comprising a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 196 or SEQ ID NO. 196 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising a CD8a hinge, a CD8a transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO 197 or SEQ ID NO 197 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:198 or SEQ ID NO:198 without amino acids 1-21.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:199 or SEQ ID NO:199 without amino acids 1-20.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO:200 or SEQ ID NO:200 without amino acids 1-18.

An exemplary CAR according to the present disclosure includes: an extracellular domain comprising an scFv comprising the amino acid sequence set forth in SEQ ID No. 132 and an extracellular spacer domain comprising an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB costimulatory signaling domain, and a CD3 zeta signaling domain. In one embodiment, the CAR comprises the amino acid sequence shown in SEQ ID NO. 201 or SEQ ID NO. 201 without amino acids 1-20.

In some embodiments, the CAR comprises the amino acid sequence of the CAR shown in Table 3, e.g., any of SEQ ID NOS 182-234, or the amino acid sequence shown in any of SEQ ID NOS 182-234 lacking the signal peptide.

Exemplary sequences of signal peptides, binding domains, extracellular spacer, transmembrane domains, and intracellular signaling domains for the CARs of the present disclosure, as well as exemplary CAR sequences are shown in table 3.

Table 3: examples of CD72 CAR and components.

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In some embodiments, the CD72 CAR of the present disclosure contains a peptide tag, such as, for example, a Myc tag (SEQ ID NO: 239). In some embodiments, the Myc tag is inserted into the CD72 CAR after the signal peptide but before the first variable region of the scFv binding domain. It will be appreciated that for the CAR sequences provided herein comprising a Myc tag, the same CAR sequence is envisaged without a Myc tag insertion sequence.

Radiation therapy includes external beam radiation therapy (e.g., conventional external beam radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy, intensity modulated radiation therapy, volume modulated arc therapy, particle therapy, proton therapy, and auger therapy), brachytherapy, systemic radioisotope therapy, intraoperative radiation therapy, or any combination thereof.

Exemplary antibodies for use in combination with the chimeric Tim compositions described herein include rituximab (rituxmab), pertuzumab (pertuzumab), trastuzumab (trastuzumab), alemtuzumab (alemtuzumab), tiuximab (Ibritumomab tiuxetan), rituximab (Brentuximab vedotin), cetuximab (cetuximab), bevacizumab (bevacizumab), acimumab (abciximab), adalimumab (adalimumab), afacib (alexaprop), baricizumab (basilizimab), belimumab (belimumab), bei Zuoluo mab (bezlotoxumab), cinanzumab (canakinumab), cetuzumab (certolizumab pegol), daclizumab (daclizumab), denouzumab (osumab), efalizumab (efuzumab), rituximab (golimumab), oxuzumab (alemtuzumab), paltuzumab (paltuzumab) and panitumumab (pauzumab).

Inhibitors of exemplary immune checkpoint molecules that may be used in conjunction with the chimeric Tim compositions described herein include checkpoint inhibitors that target PD-L1, PD-L2, CD80, CD86, B7-H3, B7-H4, HVEM, adenosine 、GAL9、VISTA、CEACAM-1、CEACAM-3、CEACAM-5、PVRL2、PD-1、CTLA-4、BTLA、KIR、LAG3、TIM3、A2aR、CD244/2B4、CD160、TIGIT、LAIR-1、PVRIG/CD112R, or any combination thereof. In certain embodiments, the immune checkpoint inhibitor may be an antibody, a peptide, an RNAi agent, or a small molecule. Antibodies specific for CTLA-4 may be ipilimumab (ipilimumab) or tremelimumab (tremelimumab). The antibody specific for PD-1 may be pidizumab (pidirizumab), nivolumab (nivolumab) or pembrolizumab (pembrolizumab). The antibody specific for PD-L1 may be dewaruzumab (durvalumab), alemtuzumab (atezolizumab), or avistuzumab (avelumab).

Exemplary chemotherapies for use in conjunction with the chimeric Tim4 receptor compositions described herein can include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule-inhibiting drugs, DNA damaging agents, antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactions (e.g., intercalators), and DNA repair inhibitors.

Chemotherapy includes nonspecific cytotoxic agents that inhibit mitosis or cell division, as well as molecular targeted therapies that prevent the growth and spread of cancer cells by targeting specific molecules (e.g., oncogenes) that are involved in tumor growth, progression, and metastasis. Exemplary non-specific chemotherapeutics for use in conjunction with the expression cassette compositions described herein can include alkylating agents, platinum-based agents, cytotoxic agents, chromatin function inhibitors, topoisomerase inhibitors, microtubule-inhibiting drugs, DNA damaging agents, antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA-interacting agents (e.g., intercalating agents), hypomethylation agents, and DNA repair inhibitors.

Examples of chemotherapeutic agents contemplated for use in the combination therapies contemplated herein include vemurafenib, dabrafenib, trametinib, cobicitinib, anastrozoleBicalutamide/>Bleomycin sulfate/>Busulfan/>Busulfan injection/>Capecitabine/>N4-pentoxycarbonyl-5-deoxy-5-fluorocytosine nucleoside, carboplatin/>Carmustine/>Chlorambucil/>Cisplatin/>Cladribine/>Cyclophosphamide (/ >)Or/>) Cytarabine, cytosine arabinoside/>Cytarabine liposome injection/>Dacarbazine/>Actinomycin (actinomycin D, cosmegan), daunorubicin hydrochloride/>Daunorubicin citrate liposome injection/>Dexamethasone, docetaxel/>Doxorubicin hydrochloride/>Etoposide/>Fludarabine phosphate/>5-Fluorouracil/>Fluotamide/>Tizacitabine, gemcitabine (difluodeoxycytidine), hydroxyurea/>Idarubicin/>Ifosfamide/>Irinotecan/>L-asparaginase/>Calcium leucovorin, melphalan/>6-Mercaptopurine/>Methotrexate/>Mitoxantrone/>Getuzumab, paclitaxel/>Phoenix (yttrium 90/MX-DTPA), penstatin, polifeprosan 20/>, containing carmustine implantTamoxifen citrate/>Teniposide/>6-Thioguanine, thiotepa, tirapazamine/>Topotecan hydrochloride/>, for injectionVinblastine/>Vincristine/>Ibrutinib, vinatoxin, crizotinib, aprepitinib, buganinib, ceritinib and vinorelbine

Exemplary alkylating agents for combination therapies contemplated herein include nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes, uratemustine (Aminouracil Uracil nitrogen/>) Nitrogen mustard/>Cyclophosphamide (/ >)Revimmune ^TM) ifosfamide/>Melamine/>Chlorambucil/>Pipobromine/> Triethylenemelamine/>Triethylenethiophosphamide, temozolomideThiotepa/>Busulfan/>Carmustine/>Lomustine/>Streptozotocin/>And dacarbazine/>Other exemplary alkylating agents for combination therapies contemplated herein include, but are not limited to, oxaliplatin/>Temozolomide (/ >)And/>) ; Actinomycin (also known as actinomycin D,/>) ; Melphalan (also known as L-PAM, L-oncolytic toxins and phenylalanine nitrogen mustard,/>) ; Altretamine (also known as altretamine (HMM),/>) ; CarmustineBendamustine/>Busulfan (/ >)And/>) ; Carboplatin/>Lomustine (also called CCNU,/>) ; Cisplatin (also known as CDDP,/>And/>) ; Chlorambucil/>Cyclophosphamide (/ >)And/>) ; Dacarbazine (also known as DTIC, DIC and imidazole carboxamide,/>) ; Altretamine (also known as altretamine (HMM),/>) ; Ifosfamide/>Prednumustine; methyl benzyl hydrazine/>Dichloromethyldiethylamine (also known as nitrogen mustard, nitrogen mediated and mechlorethamine hydrochloride,/>) ; Streptozotocin/>Thiotepa (also known as thiophosphamide, TESPA and TSPA,/>) ; Cyclophosphamide/>And bendamustine hydrochloride/>

Exemplary platinum-based agents for the combination therapies contemplated herein include carboplatin, cisplatin, oxaliplatin, nedaplatin, picoplatin, satraplatin, phenanthrlatin, and triplatin tetranitrate.

Exemplary hypomethylation agents for combination therapy include azacytidine and decitabine.

Exemplary molecular targeted inhibitors for use in conjunction with the chimeric Tim4 receptor compositions described herein include small molecules that target molecules involved in cancer cell growth and survival, including, for example, receptor tyrosine kinase inhibitors, RAF inhibitors, BCL-2 inhibitors, ABL inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4/6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PDGFRA inhibitors, and RET inhibitors.

Exemplary molecular targeted therapies include hormone antagonists, signaling inhibitors, gene expression inhibitors (e.g., translation inhibitors), apoptosis inducers, angiogenesis inhibitors (e.g., VEGF pathway inhibitors), tyrosine kinase inhibitors (e.g., EGF/EGFR pathway inhibitors), growth factor inhibitors, GTPase inhibitors, serine/threonine kinase inhibitors, transcription factor inhibitors, cancer-related driver mutation inhibitors, B-Raf inhibitors, MEK inhibitors, mTOR inhibitors, adenosine pathway inhibitors, EGFR inhibitors, PI3K inhibitors, BCL2 inhibitors, VEGFR inhibitors, MET inhibitors, MYC inhibitors, BCR-ABL inhibitors, HER2 inhibitors, H-RAS inhibitors, K-RAS inhibitors, PDGFR inhibitors, ALK inhibitors, ROS1 inhibitors, BTK inhibitors, TRK inhibitors, c-KIT inhibitors, c-MET inhibitors, CDK4/6 inhibitors, FAK inhibitors, FGFR inhibitors, FLT3 inhibitors, IDH1 inhibitors, IDH2 inhibitors, PARP inhibitors, pdgra inhibitors, and refgft inhibitors. In certain embodiments, using molecular targeted therapies includes administering molecular targeted therapies specific for a molecular target to a subject identified as having a tumor with the molecular target (e.g., driving an oncogene). In certain embodiments, the molecular target has an activating mutation. In certain embodiments, the use of chimeric Tim4 receptor modified cells in combination with a molecular targeted inhibitor increases the intensity of the anti-tumor response, the persistence of the anti-tumor response, or both. In certain embodiments, less than typical doses of molecular targeted therapies are used in combination with cells modified with chimeric Tim4 receptors.

Exemplary angiogenesis inhibitors include, but are not limited to, A6 (Angstrom Pharmaceuticals), ABT-510 (Abbott Laboratories), ABT-627 (atrasentan) (Abbott Laboratories/Xinlay), ABT-869 (Abbott Laboratories), actimid (CC 4047, pomalidomide )(Celgene Corporation)、AdGVPEDF.11D(GenVec)、ADH-1(Exherin)(Adherex Technologies)、AEE788(Novartis)、AG-013736(, axitinib) (Pfizer), AG3340 (Promaseta )(Agouron Pharmaceuticals)、AGX1053(AngioGenex)、AGX51(AngioGenex)、ALN-VSP(ALN-VSP O2)(Alnylam Pharmaceuticals)、AMG 386(Amgen)、AMG706(Amgen)、, apatinib (YN 968D 1) (Jiangsu Hengrui Medicine), AP23573 (ground pimox/MK 8669) (Ariad Pharmaceuticals), AQ4N (Novavea), ARQ 197 (ArQule), ASA404 (Novartis/Antisoma), attomode (Callisto Pharmaceuticals)、ATN-161(Attenuon)、AV-412(Aveo Pharmaceuticals)、AV-951(Aveo Pharmaceuticals)、, avastin (bevacizumab) (Genntech), AZD2171 (sildenb/Recentin) (Astrazeneca), azod 2171 (Novarena) BAY 57-9352 (Tilatinib )(Bayer)、BEZ235(Novartis)、BIBF1120(Boehringer Ingelheim Pharmaceuticals)、BIBW 2992(Boehringer Ingelheim Pharmaceuticals)、BMS-275291(Bristol-Myers Squibb)、BMS-582664( Brinib) (Bristol-Myers Squibb), BMS-690514 (Bristol-Myers Squibb), calcitriol, CCI-779 (torisel) (Wyeth), CDP-791 (ImClone Systems), cephalotaxine (homoharringtonine/HHT) (ChemGenex Therapeutics), celecoxib (celecoxib )(Pfizer)、CEP-7055(Cephalon/Sanofi)、CHIR-265(Chiron Corporation)、NGR-TNF、COL-3(Metastat)(Collagenex Pharmaceuticals)、 combretastatin (Oxigene)、CP-751、871(Figitumumab)(Pfizer)、CP-547、632(Pfizer)、CS-7017(Daiichi Sankyo Pharma)、CT-322(Angiocept)(Adnexus)、 curcumin, dalteparin (Fapamine) (Pfizer), disulfiram (antabust), E7820 (EISAI LIMITED), E7080 (EISAI LIMITED), EMD 121974 (cilengrapin) (EMD Pharmaceuticals), ENMD-1198 (EntreMed), ENMD-2076 (EntreMed), endegree (Simcere), abiturin (ImClone/Bristol-Myers Squibb)、EZN-2208(Enzon Pharmaceuticals)、EZN-2968(Enzon Pharmaceuticals)、GC1008(Genzyme)、 genistein, GSK1363089 (Foretinib) (GlaxoSmithKline), GW786034 (pazopanib) (GlaxoSmithKline), GT-111 (Vascular Biogenics Ltd.), IMC-1121B (ramucirumab )(ImClone Systems)、IMC-18F1(ImClone Systems)、IMC-3G3(ImClone LLC)、INCB007839(Incyte Corporation)、INGN 241(Introgen Therapeutics)、 Iressa (ZD 1839/gefitinib), LBH589 (Faridak/Panobinostst) (Novartis), lucentis (ranibizumab) (Genentech/Novartis), LY317615 (Enzastaurin) (ELI LILLY AND Company), macugen (pipgatanib) (Pfizer), MEDI522 (Abegrin) (MedImmune), MLN518 (tankytinib) (Millennium), neovastat (941/bikinfen) (Aeterna Zentaris), nexavar (Bayer/Onyx), NM-3 (AE GenzymeCorporation) Natobutynin (Cougar Biotechnology)、NPI-2358(Nereus Pharmaceuticals)、OSI-930(OSI)、Palomid 529(Paloma Pharmaceuticals,Inc.)、Panzem capsule (2ME2)(EntreMed)、Panzem NCD(2ME2)(EntreMed)、PF-02341066(Pfizer)、PF-04554878(Pfizer)、PI-88(Progen Industries/Medigen Biotechnology)、PKC412(Novartis)、 tea polyphenols E (green tea extract )(Polypheno E International,Inc.)、PPI-2458(Praecis Pharmaceuticals)、PTC299(PTC Therapeutics)、PTK787( W Tarani) (Novartis), PXD101 (Belgium) (CuraGen Corporation), RAD001 (everolimus) (Novartis), RAF265 (Novartis), ruigfeini (BAY 73-4506) (Bayer), revlimid (Celgene), anecortave (Alcon Research), SN38 (liposome) (Neopharm), SNS-032 (BMS-387032) (Sunesis), SOM230 (pasireotide) (Novartis), squalamine (Genoera), suramin, sotan (Pfizer), tarce (Genntech), TB-403 (Thrombogenics), tempostatin (Collard Biopharmaceuticals), tetrathiomolybdate (Sigma-Aldrich), TG100801 (TargeGen), thalidomide (Celgene Corporation), tinzaparin sodium, TKI258 (Novartis), TRC093 (Tracon Pharmaceuticals inc.), VEGF Trap (aflibercept) (Regeneron Pharmaceuticals), VEGF Trap-Eye (Regeneron Pharmaceuticals), veglin (VasGene Therapeutics), bortezomib (Millennium)、XL184(Exelixis)、XL647(Exelixis)、XL784(Exelixis)、XL820(Exelixis)、XL999(Exelixis)、ZD6474(AstraZeneca)、 vorinostat (Merck) and ZSTK474.

Exemplary B-Raf inhibitors include vemurafenib, dabrafenib and Kang Naifei B.

Exemplary MEK inhibitors include bimetanib, cobetanib, refatinib, semetanib, and tramatinib.

Exemplary BTK inhibitors include ibrutinib, brutinib (Loxo-305), tiramitinib, tolebrutinib, angstrom Wo Bulu, febrinib (GDC-0853), acartinib, vicarious brutinib (SNS-062), ONO-4059, capetinib, zebutinib (BGB-3111), HM71224, and M7583.

Exemplary TRK inhibitors include emtrictinib, lartinib, CH7057288, ONO-7579, LOXO-101, letatinib, and LOXO-195.

Exemplary c-KIT inhibitors include imatinib, sunitinib, and bunatinib.

Exemplary c-MET inhibitors include carbamazepine, crizotinib, tivantinib, onapristal bead mab, INCB28060, AMG-458, sivantinib, and terbutanib.

Exemplary CDK4/6 inhibitors include palbociclib, rebabociclib, flumazenil and troraxili.

Exemplary FAK inhibitors include difatinib, GSK2256098, BI853520, and PF-00562271.

Exemplary FGFR inhibitors include erdasatinib, pemitinib, inflitinib, luo Jiati ni, AZD4547, BGJ398, FP-1039, and ARQ 087.

Exemplary FLT-3 inhibitors include quezatinib, cladribine, ji Ruiti, midostaurin, and letatinib.

Exemplary IDH1 inhibitors include Ai Funi cloths, BAY-1436032, and AGI-5198.

Exemplary IDH2 inhibitors include azepine.

Exemplary PARP inhibitors include talazapanib, nilaparib, lu Kapa, olaparib (AZ 2281, KU 59434), veliparib (ABT 888), CEP 9722, E7016, AG014699, MK4827, BMN-673, and pamiparib (BGB-290). Exemplary PDGFRA inhibitors include imatinib, regorafenib, clarithromycin, and olamumab.

Exemplary pan-RAF inhibitors include belvarafenib, LXH254, LY3009120, INU-152, and HM95573.

Exemplary RET inhibitors include lenvatinib, aletinib, vandetanib, cabozitinib, BLU-667, and LOXO-292.

Exemplary ROS1 inhibitors include ceritinib, loratidine, emtrictinib, crizotinib, TPX-0005, and DS-6051b.

Exemplary Vascular Endothelial Growth Factor (VEGF) receptor inhibitors include, but are not limited to bevacizumabAcetirizine/>Alanine brinib (BMS-582664, (S) - ((R) -1- (4- (4-fluoro-2-methyl-1H-indol-5-yloxy) -5-methylpyrrolo [2,1-f ] [1,2,4] triazin-6-yloxy) propan-2-yl) 2-aminopropionate); sorafenib/>Pazopanib/>Sunitinib malate/>Sildenb (AZD 2171, CAS 288383-20-1); nidaminib (BIBF 1120, CAS 928326-83-4); foretinib (GSK 1363089); tiratinib (BAY 57-9352, CAS 332012-40-5); apatinib (YN 968D1, CAS 811803-05-1); imatinibPanatinib (AP 24534, CAS 943319-70-8); tivozanib (AV 951, CAS 475108-18-0); regorafenib (BAY 73-4506, cas 755037-03-7); varanib dihydrochloride (PTK 787, CAS 212141-51-0); brinib (BMS-540215,CAS 649735-46-6); vandetanib (/ >)Or AZD 6474); motsemii diphosphate (AMG 706, CAS 857876-30-3, n- (2, 3-dihydro-3, 3-dimethyl-1H-indol-6-yl) -2- [ (4-pyridylmethyl) amino ] -3-pyridinecarboxamide, as described in PCT publication No. WO 02/066470); poly Wei Tini di-lactate (TKI 258, CAS 852433-84-2); linfanib (ABT 869, CAS 796967-16-3); cabotinib (XL 184, CAS 849217-68-1); letatinib (CAS 111358-88-4); n- [5- [ [ [5- (1, 1-dimethylethyl) -2-oxazolyl ] methyl ] thio ] -2-thiazolyl ] -4-piperidinecarboxamide (BMS 3803, CAS 345627-80-7); (3R, 4R) -4-amino-1- ((4- ((3-methoxyphenyl) amino) pyrrolo [2,1-f ] [1,2,4] triazin-5-yl) methyl) piperidin-3-ol (BMS 690514); n- (3, 4-dichloro-2-fluorophenyl) -6-methoxy-7- [ [ (3 a alpha, 5 beta, 6a alpha) -octahydro-2-methylcyclopent [ c ] pyrrol-5-yl ] methoxy ] -4-quinazolinamine (XL 647, CAS 781613-23-8); 4-methyl-3- [ [ 1-methyl-6- (3-pyridinyl) -1H-pyrazolo [3,4-d ] pyrimidin-4-yl ] amino ] -N- [3- (trifluoromethyl) phenyl ] -benzamide (BHG 712, CAS 940310-85-0); and Abelmoschus/>

Exemplary EGF pathway inhibitors include, but are not limited to, tyrosine phosphorylation inhibitor 46, EKB-569, erlotinibGefitinib/>Erbitux, nimotuzumab, lapatinib/>Cetuximab (anti-EGFR mAb), ¹⁸⁸ Re-labeled nituzumab (anti-EGFR mAb), and those compounds generally and specifically disclosed in WO 97/02266, EP 0 564 409, WO 99/03854, EP 0 520 722, EP 0 566 226, EP 0 787 722, EP 0837 063, U.S. Pat. No. 5,747,498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983, and WO 96/33980. Exemplary EGFR antibodies include, but are not limited to, cetuximab/>Panitumumab/>Matuzumab (EMD-72000); trastuzumab/>Nituzumab (hR 3); zatuzumab; THERACIM H-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1). Exemplary Epidermal Growth Factor Receptor (EGFR) inhibitors include, but are not limited to erlotinib hydrochloride/>Ceritinib; b, b; ornitinib; dacatinib; icotinib; gefitinib/>N- [4- [ (3-chloro-4-fluorobenzene) amino ] -7- [ [ (3 "S") -tetrahydro-3-furanyl ] oxy ] -6-quinazolinyl ] -4 (dimethylamino) -2-butenamide,/>) ; Vandetanib/>Lapatinib/>(3R, 4R) -4-amino-1- ((4- ((3-methoxyphenyl) amino) pyrrolo [2,1-f ] [1,2,4] triazin-5-yl) methyl) piperidin-3-ol (BMS 690514); kanetinib dihydrochloride (CI-1033); 6- [4- [ (4-ethyl-1-piperazinyl) methyl ] phenyl ] -N- [ (1R) -1-phenylethyl ] -7H-pyrrolo [2,3-d ] pyrimidin-4-amine (AEE 788, CAS 497839-62-0); xylolitinib (TAK 165); pelitinib (EKB 569); afatinib (BIBW 2992); lenatinib (HKI-272); n- [4- [ [1- [ (3-fluorophenyl) methyl ] -1H-indazol-5-yl ] amino ] -5-methylpyrrolo [2,1-f ] [1,2,4] triazin-6-yl ] -carbamic acid, (3S) -3-morpholinomethyl ester (BMS 599626); n- (3, 4-dichloro-2-fluorophenyl) -6-methoxy-7- [ [ (3 a alpha, 5 beta, 6a alpha) -octahydro-2-methylcyclopent [ c ] pyrrol-5-yl ] methoxy ] -4-quinazolinamine (XL 647, CAS 781613-23-8); 4- [4- [ [ (1R) -1-phenylethyl ] amino ] -7H-pyrrolo [2,3-d ] pyrimidin-6-yl ] -phenol (PKI 166, CAS 187724-61-4); luo Xiti Ni.

Exemplary mTOR inhibitors include, but are not limited to, rapamycinAnd analogs and derivatives thereof; SDZ-RAD; tisirolimus (/ >)Also known as CCI-779); gespholimus (previously known as deferolimus, dimethyl phosphinic acid (1R, 2R, 4S) -4- [ (2R) -2[ (1R, 9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S, 35R) -1, 18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentoxy-11, 36-dioxa-4-azatricyclo [30.3.1.0 ^4,9 ] tricetyl-16,24,26,28-tetraen-12-yl ] propyl ] -2-methoxycyclohexyl, also known as AP23573 and MK8669, and described in PCT publication number WO 03/064383); everolimus (/ >)Or RAD 001); rapamycin (AY 22989,/>)) ; Sima Pi Morde (CAS 164301-51-3); (5- {2, 4-bis [ (3S) -3-methylmorpholin-4-yl ] pyrido [2,3-d ] pyrimidin-7-yl } -2-methoxyphenyl) methanol (AZD 8055); 2-amino-8- [ trans-4- (2-hydroxyethoxy) cyclohexyl ] -6- (6-methoxy-3-pyridinyl) -4-methyl-pyrido [2,3-d ] pyrimidin-7 (8H) -one (PF 04691502, CAS 1013101-36-4); and N ² - [1, 4-dioxo- [ [4- (4-oxo-8-phenyl-4H-1-benzopyran-2-yl) morpholin-4-yl ] methoxy ] butyl ] -L-arginyl glycyl-L-alpha-aspartyl-L-serine-inner salt (SF 1126, CAS 936487-67-1).

Exemplary phosphoinositide 3-kinase (PI 3K) inhibitors include, but are not limited to Du Weili sibs, iderani, 4- [2- (1H-indazol-4-yl) -6- [ [4- (methylsulfonyl) piperazin-1-yl ] methyl ] thieno [3,2-d ] pyrimidin-4-yl ] morpholine (also known as GDC 0941 and described in PCT publication nos. WO 09/036082 and WO 09/055730); 2-methyl-2- [4- [ 3-methyl-2-oxo-8- (quinolin-3-yl) -2, 3-dihydroimidazo [4,5-c ] quinolin-1-yl ] phenyl ] propionitrile (also known as BEZ 235 or NVP-BEZ 235 and described in PCT publication No. WO 06/122806); 4- (trifluoromethyl) -5- (2, 6-dimorpholin-4-yl) pyridin-2-amine (also known as BKM120 or NVP-BKM120 and described in PCT publication No. WO 2007/084786); cerclage (VX 680 or MK-0457, cas 639089-54-6); (5Z) -5- [ [4- (4-pyridinyl) -6-quinolinyl ] methylene ] -2, 4-thiazolidinedione (GSK 1059615, CAS 958852-01-2); (1E, 4S,4aR,5R,6aS,9 aR) -5- (acetoxy) -1- [ (di-2-propenylamino) methylene ] -4,4a,5, 6a, 8,9 a-octahydro-11-hydroxy-4- (methoxymethyl) -4a, 6 a-dimethyl-cyclopenta [5,6] naphtho [1,2-c ] pyran-2,7,10 (1H) -trione (PX 866, CAS 502632-66-8); and 8-phenyl-2- (morpholin-4-yl) -chromen-4-one (LY 294002, CAS 154447-36-6). Exemplary Protein Kinase B (PKB) or AKT inhibitors include, but are not limited to, 8- [4- (1-aminocyclobutyl) benzene ] -9-phenyl-1, 2, 4-thiazolo [3,4-f ] [1,6] naphthyridin-3 (2H) -one (MK-2206, CAS 1032349-93-1); pirifaxine (KRX 0401); 4-dodecyl-N-1, 3, 4-thiadiazol-2-yl-benzenesulfonamide (PHT-427, CAS 1191951-57-1); 4- [2- (4-amino-1, 2, 5-oxadiazol-3-yl) -1-ethyl-7- [ (3S) -3-piperidinylmethoxy ] -1H-imidazo [4,5-c ] pyridin-4-yl ] -2-methyl-3-butyn-2-ol (GSK 690693, CAS 937174-76-0); 8- (1-hydroxyethyl) -2-methoxy-3- [ (4-methoxyphenyl) methoxy ] -6H-dibenzo [ b, d ] pyran-6-one (palomid 529, P529, or SG-00529); tricirbine (6-amino-4-methyl-8- (. Beta. -D-ribofuranosyl) -4H, 8H-pyrrolo [4,3,2-de ] pyrimido [4,5-c ] pyridazine); (αs) - α - [ [ [5- (3-methyl-1H-indazol-5-yl) -3-pyridinyl ] oxy ] methyl ] -phenethylamine (a 675563, CAS 552325-73-2); 4- [ (4-chlorophenyl) methyl ] -1- (7H-pyrrolo [2,3-d ] pyrimidin-4-yl) -4-piperidinamine (CCT 128930, CAS 885499-61-6); 4- (4-chlorophenyl) -4- [4- (1H-pyrazol-4-yl) phenyl ] -piperidine (AT 7867, CAS 857531-00-1); and Archexin (RX-0201, CAS 663232-27-7).

In certain embodiments, the tyrosine kinase inhibitor used in combination with the chimeric Tim4 receptor-modified cells is an Anaplastic Lymphoma Kinase (ALK) inhibitor. Exemplary ALK inhibitors include crizotinib, ceritinib, aletinib, buganinib, DALANTERCEPT, emtrictinib, and loratidine.

In certain embodiments in which the chimeric Tim4 receptor modified cells are administered in combination with one or more additional therapies, the one or more additional therapies may be administered at a dose that if administered as monotherapy may be considered to be below the therapeutic dose. In such embodiments, the chimeric Tim4 receptor can provide additive or synergistic effects such that one or more additional therapies can be administered at lower doses. Combination therapy comprises administering a chimeric Tim4 receptor composition as described herein prior to (e.g., 1 day to 30 days or more prior to) additional therapy, concurrently with (on the same day) or after (e.g., 1 day to 30 days or more after) additional therapy. In certain embodiments, the chimeric Tim4 receptor modified cells are administered after administration of one or more additional therapies. In further embodiments, the cells modified with the chimeric Tim4 receptor are administered 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, or 30 days after administration of the one or more additional therapies. In still further embodiments, the cells modified with the chimeric Tim4 receptor are administered within 4 weeks, within 3 weeks, within 2 weeks, or within 1 week after administration of the one or more additional therapies. Where the one or more additional therapies involve multiple doses, the chimeric Tim4 receptor-modified cells can be administered after an initial dose of the one or more additional therapies, after a final dose of the one or more additional therapies, or between multiple doses of the one or more additional therapies.

In certain embodiments, the methods of the present disclosure comprise a depletion step. To mitigate toxicity to the subject, the step of removing the chimeric Tim4 receptor from the subject can be performed after a sufficiently long therapeutic benefit. In such embodiments, the chimeric Tim4 receptor vector can comprise an inducible suicide gene, such as iCASP, inducible Fas, or HSV-TK. Similarly, chimeric Tim4 receptor vectors can be designed to express known cell surface antigens, such as CD20 or truncated EGFR (SEQ ID NO: 38), which facilitate depletion of transduced cells by infusion of related monoclonal antibodies (mabs), such as rituximab against CD20 or cetuximab against EGFR. Alemtuzumab (Alemtuzumab) targeting CD52 present on the surface of mature lymphocytes can also be used to deplete transduced B cells, T cells, or natural killer cells.

Subjects that can be treated with the compositions and methods of the present disclosure include animals, such as humans, primates, cows, horses, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, or pigs. The subject may be male or female, and may be of any suitable age, including infant, juvenile, adolescent, adult and geriatric subjects.

Examples

Example 1: CER-T cells elicit cytotoxic effects on sensitized PTD-SER+ tumor cells

Chimeric Tim4 receptor-T cells containing TLRs are engineered to target cells that exhibit elevated levels of the cell membrane stress signaling phosphatidylserine (Ptd-Ser). Ptd-Ser is a phospholipid commonly found on the inner leaflet of plasma membrane. Upon activation of certain downstream signals (e.g., cysteine protease 3/7 activation), ptd-Ser is externalized to the outer cell membrane. It triggers a multicomponent signaling complex by participation of Ptd-Ser specific receptors on professional phagocytes/Antigen Presenting Cells (APCs), which ultimately leads to recombination of the actin cytoskeleton. Tim-4 (T cell immunoglobulin mucin-4) is one of several receptors that specifically bind to Ptd-Ser. Its expression in resident macrophages and peritoneal macrophages is associated with the clearance of apoptotic cells during normal tissue homeostasis. In Dendritic Cells (DCs), tim-4-Ptd-Ser interactions mediate antigen capture and phagocytosis for T cell cross-sensitization.

Several strategies were developed to efficiently induce or sensitize Ptd-Ser on various tumor cell lines. The sensitization step uses standard care therapeutic agents, such as targeted small molecule inhibitors, to induce cellular stress and/or apoptosis. Once induced, ptd-Ser serves as a target for chimeric Tim4 receptor-T cell engagement, activation, and cytolytic function. This two-step "sensitization and killing" strategy was demonstrated using a variety of small molecule inhibitor-chimeric Tim4 receptor-T cell product combinations, as well as combinations with engineered CAR T and TCR products.

In ovarian tumors, inhibitors of poly (ADP-ribose) polymerase (PARP), such as nilaparib, are clinically approved drugs that target the DNA damage response pathway. Responses are rarely complete and recurrence after treatment is common. To induce Ptd-Ser exposure, BRCA-2 mutated Kuramochi cell lines were treated with a therapeutic dose of the PARP inhibitor nilaparib. Brief exposure to nilaparib causes changes in membrane phospholipid symmetry in a dose-dependent manner and effectively inhibits Kuramochi cell growth (fig. 7A). The addition of chimeric Tim4 receptor pCTX133 (Tim 4 binding domain-TLR 2 signaling domain-CD 3z signaling domain) enhanced the efficacy of nilaparib in vitro at a low effector to target ratio (1:1), demonstrating the ability of pCTX133 to elicit direct cytotoxic effects on target cells (fig. 7B) as compared to transduced controls.

In mantle cell lymphomas, inhibitors of Bruton's Tyrosine Kinase (BTK), such as ibrutinib, are clinically approved drugs targeting pro-survival kinases. Responses are rarely complete and recurrence after treatment is common. Treatment JeKo-1 MCL with ibrutinib exposes Ptd-Ser as determined by immunohistochemistry using recombinant murine Tim4 protein (fig. 8A). The JeKo-1 set of cell lymphoma cell lines were treated with 25uM ibrutinib for 24 hours, then the drug was washed away and co-cultured with 0.5uM ibrutinib and chimeric Tim4 receptor pCTX136 (Tim 4 binding domain-CD 28 signaling domain-CD 3z signaling domain) or control cells at an E: T ratio of 3:1, 2:1 or 1:1. Co-culture with chimeric Tim4 receptor cells induced near elimination of JeKo-1 target cells compared to ibrutinib treatment or treatment with control T cells (FIG. 8B).

Chimeric Tim-4 receptors with TLR2 or TLR8 intracellular signaling domains, or CD28 or CD3 zeta intracellular signaling domains were tested for their ability to promote tumor cell acquisition and elicit cytotoxicity and APC-like functions. To assess antigen acquisition, target cells were co-cultured with chimeric Tim-4 receptor T cells and assessed by Transmission Electron Microscopy (TEM) or flow cytometry. Target cells were treated with small molecule inhibitors to induce Ptd-Ser extravasation and lysosomal uptake was assessed using a pH indicator dye (borod Red).

Alternative sensitization and killing therapeutic strategies combine chimeric Tim4 receptor-T cells containing TLRs with chimeric antigen receptor-T (CAR) -T cells. This combination approach utilizes CAR to specifically target tumor cells, resulting in up-regulation of Ptd-Ser. CD19 CAR-T cell products (anti-CD 19 scFv-CD28 costimulatory signaling domain-CD 3 zeta signaling domain; "1928z CAR") rapidly induced Ptd-Ser on CD19+ Mantle Cell Lymphoma (MCL) cells in a dose-dependent manner (FIG. 5A). In co-culture studies, the combination of 1928z CAR-T cells (pCTX 184) and pCTX131 (Tim 4-TLR8-CD3 z) showed enhanced potency as measured by incucyte and FACS. 1928z CAR T cells were combined at low effector: target ratio using pCTX (Tim 4-TLR8-CD3 z) cells at multiple CAR: CER ratios (fig. 5B, fig. 6A). pCTX156 is a truncated EGFR (EGFR) control. An increase in inflammatory cytokines such as IFN-gamma was observed from the supernatant, consistent with the observed increase in cytolytic function (FIG. 5C). Furthermore, in co-culture studies, the combination of 1928z CAR-T cells and pCTX (Tim 4-TLR8-CD3 z) showed enhanced induction of cleaved cysteine proteases in target cells as measured by incucyte. pCTX131 (Tim 4-TLR8-CD3 z) cells were used to combine 1928z CAR T cells at low effector: target ratio at multiple CAR: CER ratios (fig. 6B).

Thus, this example underscores that chimeric Tim4 receptor-T cells containing pCTX and pCTX131 (TLR 2 and TLR 8) can elicit cytolytic activity against primed cell surface Ptd-Ser expressing solid tumors and hematologic target cell lines, and enhance small molecule and CAR-based therapies.

Example 2: chimeric TIM4 receptor-T cell mediated antigen capture and presentation

Activated T cells have been demonstrated to process and present Ag because they express class II molecules, display antigens on the cell surface, and can deliver costimulatory signals ¹⁰ to other T cells. However, unlike professional APCs, T cells are limited ⁹ by the inability to capture soluble antigens efficiently. In contrast, APCs utilize constitutively expressed Ag uptake receptors to capture and phagocytose antigens for subsequent degradation and MHC loading ^{12 13}. In the presence of surface receptors that bind Ag with high affinity, the capture efficiency of soluble antigens can be as high as 10 ³ times ¹⁴.

In Dendritic Cells (DCs), tim-4-Ptd-Ser interactions mediate capture, phagocytosis and concentration of antigens, allowing DCs to efficiently present antigens to T cells. Indeed, tim-4 receptors block activation of lesion-specific cd8+ T cells and promote tumor progression ¹² in preclinical NSCLC models. Furthermore, gene expression profiles showed down-regulation of Tim-4 expression in late stage tumor cells, consistent with reduced antigen uptake, presentation and T cell activation.

The experimental results provided herein demonstrate that T cells can be redirected to antigen presenting T cells for immunotherapy by enhancing their antigen uptake, capture and co-stimulatory capabilities. Fusion of the human Tim-4 phagocyte uptake receptor with intracellular signal sequences that drive antigen uptake, antigen processing and presentation increases the enhanced APC capacity of T cells. The modular design of chimeric Tim4 receptors incorporates intracellular domains that drive multicomponent signaling complexes (such as cd3ζ, CD28, 4-1BB, ITAM, and TLR signaling) to induce cell activation, cytolytic function, secretion of cytokines and chemokines, adhesion, and upregulation of co-stimulatory molecules, and to mediate antigen degradation processes ¹⁵ required for potent T cell activation.

Chimeric Tim4 receptor-T cells were tested to determine if they could capture and present soluble antigens and trigger activation and proliferation of recombinant E7 restricted T cell clones in a co-culture system. Expression of Tim4 was demonstrated for the first time by flow cytometry on transduced CER T cells. Tim4 binding domain-CD 28 transmembrane region-CD 28 signaling domain-CD 3z signaling domain (CTX 247, also known as CER 1161) and Tim4 binding domain-CD 28 signaling domain-CD 3z signaling domain-TLR 2 signaling domain (CTX 1107, also known as CER1107 or CER1236-SEQ ID NO: 19) stained Tim4 and EGFR (a transduction marker encoded on each vector). CER1107 and CER1236 have the same amino acid sequence but different carrier backbones. Tim4 expression was observed on CTX247 or CTX1107 transduced cells, but not on mock transduced control cells (FIG. 4). The E7-restricted TCR targets the E7 protein from HPV16 and has the TCR alpha and TCR beta chain sequences provided in SEQ ID NO 241. E7 TCRs are proliferated by MHC class I in response to APC pulsed with E7 peptide. For autologous APCs, cd4+ and cd8+ chimeric Tim4 receptor T cell products transduced with different Tim-4 chimeric receptors were pulsed with a 15-mer peptide pool containing 11 amino acid overlaps with the E7 protein from HPV16 or vectors. Chimeric Tim4 receptor T cells were pulsed with E7 peptide at 37 ℃ for 4 hours and tested for their ability to trigger E7-specific activation and proliferation. After 24 hours of co-culture with chimeric Tim4 receptor-T cell products, the E7-TCR cell surface activation marker response was assessed by flow cytometry. After another 5 days of co-culture, proliferation response was assessed by CELL TRACE (CT) Violet dilution.

FIGS. 1B-1D show pCTX1107 (Tim 4 binding domain-CD 28 intracellular signaling domain-CD 3 zeta intracellular signaling domain-TLR 2 intracellular signaling domain; SEQ ID NO: 19) CER T cells do have stimulatory effects on E7-specific T cells, whereas pCTX247 (Tim 4 binding domain-CD 28 intracellular signaling domain-CD 3 zeta intracellular signaling domain) or non-transduced T cells have NO stimulatory effects, even when high concentrations of E7 peptide pulses are used. In these experiments, the only difference in construct design between pCTX247 and pCTX1107 was the addition of TLR-2 intracellular sequences (fig. 1A), suggesting a role for TLR signaling in T cell presentation of amplified soluble antigen and triggering E7 TCR. Proliferation induced by CER T cells is HLA dependent, confirming the role of antigen presentation in CER-mediated activity.

PCTX1107 proof that 1107 is a strong stimulus for E7 specific activation was also observed in CD25 and CD69 upregulation as measured by flow cytometry (fig. 2). After 24 hours of co-culture, E7-TCR-containing T cells expressed both activation markers more frequently (FIG. 2). After 24 hours of co-culture with chimeric Tim4 receptor-T, the E7 TCR-T cell surface activation markers CD25 and CD69 were upregulated by 41.2% and 23.1%, respectively, relative to the control, and the percentage of dividing E7-TCR-T cells after 6 days was 44% for Tim4/CD28/CD3z/TLR2 chimeric Tim4 receptor T cells, relative to the control.

CELL TRACE Violet-labeled E7-specific TCR T cells were incubated with non-transduced T cells (UTs), T cells transduced with either a Tim4-CD28-CD3z construct (CTX 247) or a Tim4-CD28-CD3z-TLR2 construct (CTX 1107) and JeKo-1 cells at a ratio of 1:2:2 in the presence of a 15-mer peptide pool (100 ng of each peptide) derived from HPV 16E 7 protein with 11 amino acid overlap for 4 days. The duplicate cultures were incubated with HLA A A, B, C blocking antibodies (clone W6/32) or matched mouse IgG2a antibodies for the duration of the incubation. Figure 10 shows the percentage of E7 TCR cells cultured in live cells as determined by flow cytometry based on staining of mouse tcrb+ cells. Activation of E7-specific TCR T cells mediated by chimeric Tim4 receptor antigen presentation is blocked by anti-HLA-I antibodies.

Example 3: transfection of T cells with chimeric TIM4 receptor

Chimeric Tim4 receptors pCTX, pCTX1161, pCTX1189, pCTX1184, pCTX1163, pCTX1162, pCTX1190, pCTX1186, pCTX1187, pCTX1164, pCTX1185 and pCTX1165 (see table 8) were transfected into Jurkat T lymphocyte lines (fig. 9A-9B).

Example 4: enhanced antigen capture, antigen presenting cell-like function and cytotoxic response in the case of chimeric TIM4 receptor T cells

The method comprises the following steps:

Healthy donor T cells were used to generate chimeric Tim4 receptor T cells (also known as CER T cells) containing Tim-4 receptors fused to various transmembrane and intracellular signaling domains (fig. 11). CER1234 has the amino acid sequence shown in SEQ ID NO. 18. CER1107 (also referred to herein as CTX1107 or CER 1236) has the amino acid sequence shown in SEQ ID NO: 19. CER1107 and CER1236 have the same amino acid sequence, but their lentiviral backbones are different. CER247 and CER1161 (Tim 4 binding domain-CD 28tm-CD28icd-CD3zICD, SEQ ID NO: 21) have identical amino acid sequences, but their lentiviral backbones and promoter systems are different.

At day 6 after transduction of Tim-4 expression, CER T cell products (CD 45RA, CCR7 and CD4/CD8 ratios) were characterized, all as measured by Flow Cytometry (FC) using commercially available fluorochrome conjugated antibodies.

Induction of surface phosphatidylserine expression in REC-1 and JeKo-1 Mantle Cell Lymphoma (MCL) cell lines treated with ibrutinib was measured by FC using histidine-tagged recombinant murine Tim-4; secondary detection was performed using anti-histidine antibodies.

Phagocytosis was assessed in a co-culture assay of REC-1 cells with CER T cells. REC-1 cells were pretreated with 10. Mu.M ibrutinib for 24 hours, labeled with pHrodo red, and cultured with either non-transduced T cells or CER T cells at an effector to target (E: T) ratio of 0.1 to 1. After 16 or 40 hours of co-culture, the percentage of pHrodo red positive cells was determined by flow cytometry. Localization of tumor fragments in CER T cell lysosomal compartments was evaluated in a separate experiment using REC-1 cells labeled with borod green; after co-culture, all cells were labeled with Lyso-tracker red. Phagocytosis was visualized by fluorescence microscopy. Post-mortem assays using one-way ANOVA and Dunnett were compared to non-transduced T cells.

In the co-culture assay with REC-1 cells, use was made ofThe live-cell assay system was used to evaluate the cytotoxic function of CER T cells. REC-1 cells engineered to constitutively express mCherry (REC-1 mCherry) were pretreated with 10. Mu.M ibrutinib or vehicle (20% 2-OH. Beta. -cyclodextrin) for 24 hours. Ibrutinib is washed away prior to co-culture with CER T cells. CER T cells (expanded 6 days post transduction) were co-cultured with Jing Yilu-tenib-pretreated REC-1 cells in the presence of 0.5. Mu.M ibrutinib or vehicle at a 1:1 E:T ratio. The total fluorescence of REC-1mCherry cells decreased with cytotoxicity.

The T cell activation markers programmed death-1 (PD-1) and 4-1BB were evaluated on live CD3+ T cells by flow cytometry using commercially available fluorochrome conjugated antibodies. REC-1mCherry cells were pre-treated with 10. Mu.M ibrutinib or vehicle for 24 hours; after washing out, co-cultivation was started with 0.5 μm ibrutinib or vehicle. The assay was performed 96 hours after the start of co-culture of CER T cells with REC-1 cells.

In the cytotoxicity assays described above, cytokine induction was assessed by a multiparameter ELISA using conditioned supernatants harvested after 96 hours.

Antigen Presenting Cell (APC) -like function of CER T cells was evaluated as follows (fig. 1A). CELL TRACE Violet (CTV) labeled E7T cell receptor (TCR) T cells were cultured with non-transduced T cells or CER T cells and JeKo-1 cells at a ratio of 1:2:2 in the presence of a 15 mer peptide pool (100 ng of each peptide) derived from HPV 16E 7 protein with 11 amino acid overlap for 6 days. To demonstrate the effect of Major Histocompatibility Complex (MHC) class I antigen presentation, cultures as described above were incubated with mouse anti-Human Leukocyte Antigen (HLA) class I (A, B and C) blocking antibodies (clone W6/32) or matched mouse IgG2a antibodies for 4 days prior to analysis. Proliferation was measured by dye dilution of CTV.

Results:

CER is expressed by T cells (table 4). A high proportion of CER T cells express the engineered Tim-4 receptor. After transduction and expansion of CER T cells, the CD4/CD8 ratio was similar relative to non-transduced T cells. CER T cells exhibit major central memory and effector memory phenotypes as measured by CCR7 and CD45 RA.

Table 4: CER T cell characteristics (as measured on day 6 post transduction)

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Ibrutinib induces phosphatidylserine exposure in REC-1 and JEKO-1 cells

Ibrutinib (0.05 to 50 μm for 24 to 48 hours) induced PS exposure was measured by flow cytometry (fig. 12A-12B). REC-1 cells are more sensitive to ibrutinib-induced phosphatidylserine exposure than JeKo-1 cells.

CER1161 and CER 1234T cells showed phagocytic function

CER-1161 and CER-1234T cells increased phagocytosis of pHrodo red+ target cells relative to non-transduced T cells after 16 and 40 hours of co-culture as measured by flow cytometry and fluorescence microscopy (FIGS. 13A-13D). CER constructs containing intracellular signaling domains (i.e., CD28, cd3ζ, TLR 2) can enhance cytolytic function, resulting in enhanced phagocytosis.

In combination with ibrutinib, CER 1234T cells showed cytotoxic activity

Tumor cell killing was enhanced by a combination of CER-1234T cells and ibrutinib (pretreatment, co-culture, or both; fig. 14A and 14B). Co-culture with 0.5 μm ibrutinib reflects the concentration (Cmax) following standard dosing (560 mg, once daily) and has similar cytotoxic effects as pre-treatment alone or pre-treatment and co-culture with ibrutinib. CER-1183T cells (without intracellular signaling domain) did not show additive effects with ibrutinib. CER-1234T cells alone exhibit limited cytotoxic activity.

CER 1234T cells express T cell-activated cell surface markers when co-cultured with ibrutinib-treated REC-1 cells

Co-culture of CER-1234T cells and target REC-1 cells in the presence of 0.5. Mu.M ibrutinib increased the expression of PD-1 and 4-1BB on the surface of T cells (FIGS. 15A and B). PD-1 and 4-1BB were not increased in co-cultures containing CER-1183T cells lacking the intracellular signaling domain.

Induction of cytokine expression in Co-cultures of CER 1234T cells and ibrutinib-treated REC1 cells

The following classes of cytokines were induced in co-cultures of CER-1234T cells and target REC-1 cells in the presence of 0.5 μm ibrutinib (fig. 16A to 16H): t1-tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and Interleukin (IL) -10; T2-IL-5; effector-granzyme B. Cytokine expression was not induced in co-cultures containing CER-1183T cells containing Tim-4 but lacking the intracellular signaling domain. IL-2 (steady state), IL-4 (T2) and IL-6 (inflammatory) are nominally increased. IL-12p70 (APC), IL-17A (inflammatory) and IL-1. Beta. (inflammatory) are at the detection limit under all co-culture conditions.

Example 5: chimeric TIM4 receptor T cell therapies elicit phosphatidylserine-dependent cytotoxicity and antigen presenting cell-like functions and work in concert with the BTK inhibitors of the arms to combat hematologic malignancies

The tumor microenvironment largely suppresses the immune response, in part due to dysfunction of antigen presentation. While activated conventional αβ T cells are nominally capable of processing and displaying antigens, they have limited antigen presentation capacity due to inefficient antigen capture. Chimeric Tim4 receptors promote Antigen Presenting Cell (APC) like functions and confer a target-dependent cytotoxic response. Phosphatidylserine (PS) receptor Tim-4, which plays a central role in the cross presentation of dendritic cell subsets, fuses with the innate signaling domain to drive tumor antigen uptake and enhance APC-like function. APC-like and cytotoxic responses were tested using a chimeric Tim4 receptor T cell versus Mantle Cell Lymphoma (MCL) cell line engineered with a Tim-4 binding domain fused to a toll/interleukin-1 (TIR) domain and T cell-derived signaling domains CD28 and CD3 zeta.

Chimeric Tim-4 receptor constructs are designed to enhance target-dependent phagocytosis and cytotoxicity. Tumor fragment uptake, cytotoxicity, cytokine secretion (GrB, IFN-gamma and TNF-alpha) and APC-like activity were assessed in vitro using an engineered TMEM30a phosphoinvertase knockout JeKo-1 lymphoma cell line with constitutive phosphatidylserine exposure (JeKo-1 PS ⁺). JeKo-1 PS ⁺ cells were labeled with a pH sensitive pHrodo ^TM dye to quantify tumor cell uptake. After co-culturing the chimeric Tim4 receptor-T cells with JeKo PS ⁺ cells and HPV-derived peptides, the antigen-displaying capacity of the chimeric Tim4 receptor-T cells was tested by assessing activation and proliferation of autologous HPV E7 TCR T cells. Finally, the antitumor activity of chimeric Tim4 receptor T cells was examined in a xenograft MCL model. Statistical analysis was performed using two-way ANOVA.

T cells expressing CER-1234 (SEQ ID NO: 18) and CER-1236 (SEQ ID NO: 19) are engineered to readily express Tim-4 on the cell surface and the TLR2 TIR signaling domain is oriented differently relative to the T cell-derived signaling domains CD28 and CD3 zeta. Both CER1234 and CER 1236T cells showed increased uptake of JeKo-1 PS ⁺ tumor cell fragments (p < 0.001) when co-cultured in vitro. Internalization of the pHrodo ⁺ JeKo-1 fragment was dependent on Phosphatidylserine (PS) binding, and treatment with cytochalasin D (an inhibitor of actin polymerization) or bafilomycin A (a lysosomal inhibitor) blocked tumor cell fragment uptake (p <0.0001 and p < 0.0001). In REC-1 cells primed with BTK inhibitor (BTKi), the CER-1234 internalized fragment is localized to the lysosomal compartment.

After co-culture with JeKo-1 PS ⁺ cells in vitro, both CER T cell constructs showed cytotoxic function, with CER-1234 eliminating 90% of the target at a low effector to target (E: T) ratio within 96 hours. BTKi treatment in combination with CER-1234 exhibited >90% cytotoxicity and produced Th1 cytokines (GrB, IFN-gamma and TNF-alpha). Finally, the APC-like activity of CER T cells was assessed in vitro. HPV E7 TCR T cells showed a significant increase in proliferation at 72 hours (p < 0.05) compared to the control, indicating that Tim-4 CER T cells were able to process, display and trigger proliferation of antigen-specific T cells. In vivo, CER T cells reduced tumor burden compared to controls in xenograft models of MCL, with no significant morbidity observed.

CER1234 and/or CER 1236T cells overcome the antigen presentation defect in the tumor microenvironment by enhancing antigen acquisition, APC-like function and cytotoxic response. These CER T cells exhibit the combined function in vitro, act synergistically with BTK inhibition, and mediate significant anti-tumor effects in vivo against clinically relevant models of mantle cell lymphoma.

The ability of T cell antigens to capture and present, combined with inducible and target-specific cytotoxic functions in single T cells, suggests the potential for secondary immune responses via activation and enhancement of endogenous anti-tumor immunity.

Method of

Healthy donor T cells were used to generate CER T cells containing T cell immunoglobulins and mucin-domain-4 (TIM-4) containing receptors fused to various transmembrane and intracellular signaling domains (see Table 5 below).

Table 5: CER construct design

CER T cell activation was assessed by measuring induction of IFN- γ by plate-based and cell-based assays. Single induction plate assay: different concentrations of Phosphatidylserine (PS) or Phosphatidylethanolamine (PE), a control membrane phospholipid not recognized by wild-type TIM-4 (wtTIM-4), were coated onto high-binding tissue culture plates. CER T cells were added and IFN- γ levels were measured by ELISA 24 hours after ligand exposure. EC50 concentrations (agonist versus response-variable slope (4 parameters)) were determined by nonlinear regression. Induction as determined by recursive stimulation plate: CER T cells were added to phosphatidylserine coated plates (2.5-20. Mu.g/mL PS coating). Five days later, CER T cells were transferred to uncoated plates containing fresh T cell optimized medium for 24 hours. Then, CER T cells were transferred again onto fresh PS-coated plates. Supernatants were collected at different time points (24, 72 and 120 hours) during restimulation to assess IFN- γ induction by ELISA. Cell-based assays: cytokine induction was assessed by a multiparameter ELISA using conditioned supernatants harvested after 120 hours from the cytotoxicity assays described below.

In a co-culture assay with Jeko1 TMEM30A ^-/- cells (by knocking out TMEM30A gene, a reverse enzyme partner required for Phosphatidylserine (PS) internalization) or REC-1MCL cells, use was made ofThe live-cell assay system was used to evaluate the cytotoxic function of CER T cells. REC-1MCL cells engineered to constitutively express mCherry were pretreated with 100nM ibrutinib or no drug for 96 hours. CER T cells (6 days post transduction expansion) were co-cultured with A2780 cells in the presence of ibrutinib at the same concentration in an effector to target (E: T) ratio of 0.25:1 in T cell optimized medium containing IL-2, IL-7 and IL-15.

Expression of TIM-4, CD45RA and CCR7 and CD4/CD8 ratio were assessed by flow cytometry using commercially available fluorochrome conjugated antibodies. Proliferation was measured by flow cytometry using precision counting beads to determine the absolute count of live T cells at the end of co-culture. T cell proliferation is reported as the fold expansion of T cell counts at 120 hours relative to T cell counts at 0 hours.

After 48 hours of co-culture with phosphatidylserine positive E7 oncoprotein positive cells, CER T cells were evaluated for Antigen Presenting Cell (APC) -like function. T cells were isolated and co-cultured with CELL TRACE violet-labeled E7 TCR T cells for 4 days. Flow cytometry was used to analyze the activation marker HLA-DR on E7 TCR T cells. Phosphatidylserine positive SCC152 squamous cell carcinoma cells were engineered by knocking out TMEM30A gene, a reverse enzyme chaperone required for PS internalization. The role of HLA-I in APC-like function is determined by conducting the same experiment in the presence of HLA-I blocking or corresponding isotype antibodies.

Results:

CER (CER 1234 and CER 1236) was constructed and expressed in T cells according to table 5. CER T cells readily expressed TIM-4 on transduced T cells 6 days post transduction (fig. 17A). After transduction and expansion of CER T cells, the CD4 to CD8 ratio was similar relative to non-transduced T cells. CER1234 or CER 1236T cells exhibited major initial and central memory phenotypes as measured by CCR7 and CD45RA expression (fig. 18).

CER1234 or CER 1236T cells specifically produced IFN- γ in a dose-dependent manner in response to binding to Phosphatidylserine (PS) but not to Phosphatidylethanolamine (PE) (fig. 19A-19B). wtTIM-4 lacking intracellular T cell signaling moiety did not induce IFN-gamma upon phosphatidylserine stimulation. CER-1236T cells elicited repeated IFN- γ responses after 3 consecutive rounds of exposure to phosphatidylserine (fig. 19C). The assay design is shown in fig. 43. The ability of CER-1236T cells to become reactivated upon in vitro exposure to additional antigen suggests a durable effect in vivo.

FACS and imaging-based detection of phagocytosis and endocytosis of pHrodo dye-labeled tumor cells (Jeko 1-TMEM30A ^-/- cells) by CER1234T cells is shown in FIG. 20.

CER 1236T cells showed increased phagocytosis of Jeko1 TMEM30a ^-/- cells (figure 21). CER 1236T cells have enhanced phagocytosis relative to wtTIM-4T cells. Phagocytosis is blocked by mutations in the TIM-4 phosphatidylserine binding domain (CER 1250) or by inhibitors of actin polymerization. CER-1250 (Tim-4 binding mutant/CD 28/TLR2/CD3z; SEQ ID NO: 24) has the same intracellular signaling domain as CER1236 but has a mutated IgV domain with AAAA substitution to eliminate phosphatidylserine binding. Higher uptake frequency and magnitude (phagocytosis index) were observed by CER-1236T cells compared to wtTim-4, CER1250, and CER1236+ actin polymerization inhibitors.

CER1234 or CER 1236T cells effectively eliminated Jeko1 TMEM30a ^-/- cells in the co-culture assay (fig. 22).

CER 1236T cells act synergistically with ibrutinib to enhance killing of REC-1 set of cell lymphoma cells. The BTK pathway inhibits triggering cellular stress and membrane phosphatidylserine exposure. Once primed, CER 1236T cells eliminate tumor cells (FIG. 23). CER 1236T cells had enhanced and more complete cytotoxicity on ibrutinib-treated REC-1MCL cells, but no cytotoxicity on untreated REC-1MCL cells (fig. 38). Some CER 1236T cytotoxicity was observed against untreated REC-1 cells, consistent with constitutive phosphatidylserine exposure measured in this cell line. CER1251 (Tim-4 binding mutant/CD 28/CD3z/TLR2; SEQ ID NO: 25) has the same intracellular signaling domain and orientation as CER1236 but has a mutated IgV domain with an AAAA substitution to cancel phosphatidylserine binding.

Consistent with the synergistic cytotoxicity, CER1234 cells or CER1236 cells plus inhibition of BTK with ibrutinib showed inducible cytokine production (fig. 24 and 37).

CER1236T cells exhibit antigen presenting cell-like functions in an in vitro system. In the E7 HLA2 restricted TCR system, CER1236-T cells support greater activation of autologous E7TCR T cells when compared to non-transduced T cells or anti-CD 19 CAR T cells, suggesting enhanced E7 antigen presentation by CER-1236T cells (fig. 25A). CER1236T cell-induced E7TCR activation was dependent on antigen presentation via HLA class I system (fig. 25B). CER1236T cells can acquire antigen, a function that is considered to be a barrier to its ability to process and present antigen to the naive T cells. Engineered Antigen Presenting Cell (APC) functions of CER1236T cells may lead to initiation of a secondary immune response in vivo.

CER 1236T cells showed anti-tumor effect against Jeko1 TMEM30a ^-/- cell subcutaneous tumor grafts in a mantle cell lymphoma mouse model (fig. 26).

Example 6: t cell phenotype on chimeric TIM4 receptor-transduced T cells co-cultured with PARP inhibitors and target ovarian cancer cells

For the assays described herein, a2780 (human ovarian cancer cell line) cells were plated, allowed to adhere, and treated wotj PARP inhibitor (PARPi) at an effective concentration (EC 25), 0.5 μm nilaparib, or 1.5 μm olaparib that killed 25% of the cells on day-2. On day 0, the medium was changed and donor T cells from three subjects transduced with CER-1234, CER-1236 or control CER-1183 (FIG. 28) were added to A2780 cells treated with PARPi at various effector to target ratios to assess chimeric Tim4 receptor-T cell activation, cytokine production, proliferation and cytotoxic response. Immunophenotyping and extensive supernatant analysis of chimeric Tim4 receptor-T cells was performed on days 2 and 5 after the start of co-culture.

After 120 hours of co-culture, CCR7 expression was measured by measuring% ccr7+ cells in tim4+ T cells (fig. 29A-29C). Higher CCR7 expression was observed on CER-1234 and CER-1236 expressing T cells co-cultured with olaparib treated a2780 cells. CCR7 is expressed at high levels on primary and central memory T cells and enables a subset of steady state T cells to be recycled and returned to T cell areas in lymphoid organs, such as the spleen and the white marrow areas of lymph nodes. CCR7 expressing T cells may reflect a T cell population in an early differentiation state.

The CD4/CD8 ratio was measured after 120 hours of co-culture with PARPi-treated A280 cells (FIGS. 30A-30C). CER-1236 transduced T cells had a higher CD4/CD8 ratio in both donor 38 and donor 41 samples compared to T cells transduced with CER-1234 or control CER-1183.

Example 7: in ovarian cancer models, TIM4 chimeric receptor-T cells elicit phosphatidylserine dependent cytotoxicity and innate-like functions and act synergistically with PARP inhibitors from the collection

In the PARP-sensitive ovarian cancer model, CER T cells were evaluated in combination with a small molecule inhibitor of the poly (ADP-ribose) polymerase (PARP) enzyme in batches.

Phosphatidylserine exposure of a2780 ovarian cancer cells after treatment with the small molecule PARP inhibitor nilaparib or olaparib was assessed by annexin v staining to determine the appropriate drug dose. The phosphatidylserine targeted Tim-4CER construct was engineered designed to enhance cytotoxicity and target-dependent tumor cell fragment uptake. CER T cells were designed as follows: CER-1234 (Tim-4/CD 28/TLR2/CD3z; SEQ ID NO: 18); CER-1236 (Tim-4/CD 28/CD3z/TLR2; SEQ ID NO: 19); and CER-1250 (Tim-4 binding mutant/CD 28/TLR2/CD3z; SEQ ID NO: 24). CER-1250 has the same intracellular signaling domain as CER1236 but has a mutated IgV domain with AAAA substitution to eliminate phosphatidylserine binding. Nilapatinib (0.5. Mu.M) and Olaparib (1.5. Mu.M) were used to expose phosphatidylserine on the surface of A2780 cells for CER T cell recognition and to evaluate cytotoxicity, T cell activation and cytokine response. Antigen uptake by CER-1236T cells was modeled with squamous cell carcinoma (SCC 152) TMEM30A phosphoinvertase knockout cells expressing the human papillomavirus E7 antigen. These cells are constitutively exposed to the outer surface phosphatidylserine. E7 antigen presentation by CER-1236T cells was determined by surface expression of HLA-DR, a marker of T cell activation, on autologous HLA-A2 restricted E7 TCR T cells.

PARP inhibitors induced phosphatidylserine exposure on the extracellular surface in living a2780 cells at sub-therapeutic doses 96 hours after treatment. 23% of the a2780 cells were cleared after the addition of nilaparib and 34% of the cells were cleared after the addition of olaparib, compared to untreated. CER-1234 or CER-1236 eliminated A2780 cells by 39% or 19%, respectively. Nilapatinib works synergistically with CER-1234 or CER-1236 to eliminate 58% or 81% of A2780 cells after 5 days of co-culture, whereas Olaparib either eliminates 66% of the target in combination with CER-1234 or 73% of the target in combination with CER-1236. At 96 hours, interferon-gamma was increased by a factor of 7 and granzyme B was increased by a factor of 5-7 for either drug in combination with CER-1234 as compared to CER-1234 without drug. CER-1236T cells exposed to TMEM30A ^- SCC152 cancer cells increased the activation status of E7 TCR T cells 5-fold relative to CER-1236T cells alone, demonstrating partial phagocytosis, antigen processing and presentation of HLA-A2 cognate peptides.

CER1234 and CER 1236T cells exhibit adaptive and innate functions. CER1234 and CER 1236T cells have shown cytolytic activity against a2780 ovarian cancer cells treated with sub-therapeutic doses of PARP inhibitors. CER1236 showed enhanced APC-like function in HPV E7 model. The combined function of CER T cell products provides a promising clinical application as a treatment option for patients with ovarian cancer who receive a batch of PARP inhibitors.

Method of

Induction of surface phosphatidylserine expression on a2780 ovarian cells treated with PARP inhibitors olaparib and nilaparib was measured by Flow Cytometry (FC) using annexin V.

Healthy donor T cells were used to generate CER T cells containing T cell immunoglobulins fused to various transmembrane and intracellular signaling domains and an acceptor containing mucin domain-4 (TIM-4) (see Table 5).

In the co-culture assay with A2780, use was made ofThe live-cell assay system was used to evaluate the cytotoxic function of CER T cells. A2780 cells engineered to constitutively express mCherry were pretreated with 1.5 μm olaparib, 0.5 μm nilaparib, or no drug for 96 hours. CER T cells (6 days post transduction expansion) in T cell optimized medium containing IL-2, IL-7 and IL-15 were co-cultured with A2780 cells in the presence of the same concentrations of Olaparib or Nilapatinib at a ratio of effector to target (E: T) of 1:2.

Results:

Sub-therapeutic doses of nilaparib and olaparib induced phosphatidylserine exposure on living a2780 cells (fig. 31A-31B). Based on these curves, as shown by the dashed lines, a dose of 1.5 μm was selected for olaparib and a dose of 0.5 μm was selected for nilaparib for combination therapy with CER T cells.

CER constructs were designed as single chain chimeric proteins according to table 5. The phenotype of CER T cells is generally consistent between 2 donors (fig. 32). A high proportion of CER T cells express the engineered Tim-4 receptor. After transduction and expansion of CER T cells, the CD4 to CD8 ratio was similar relative to non-transduced T cells. CER1234 or CER 1236T cells exhibited major initial and central memory phenotypes as measured by CCR7 and CD45RA expression.

CER1234 or CER 1236T cells produced a concentration-dependent IFN- γ response when stimulated with phosphatidylserine (fig. 33A-33B). CER T cells showed target specific cytokine induction for phosphatidylserine but not for phosphatidylethanolamine. Saturation of IFN-gamma induction by CER T cells occurs at 5 to 10. Mu.g/mL phosphatidylserine. wtTIM-4 (which lacks the intracellular T cell signaling moiety) does not induce IFN-gamma upon phosphatidylserine stimulation. CER-1236T cells elicited repeated IFN- γ responses after 3 consecutive rounds of exposure to phosphatidylserine (fig. 33C). The ability of CER-1236T cells to become reactivated upon in vitro exposure to additional antigen suggests a durable effect in vivo.

CER1234 or CER 1236T cells showed synergistic cytotoxic activity against a2780 cells with nilaparib and olaparib. Tumor cell killing was enhanced by combinations of CER-1236 or CER-1234T cells with nilaparib or olaparib (FIGS. 34A-34B). wtTIM-4T cells (without intracellular signaling domain) showed no additive effect with either Nilapatinib or Olaparib. CER-1236 and CER-1234T cells alone exhibit limited cytotoxic activity. CER-1234 and CER-1236 proliferated in co-cultures treated with PARPi compared to co-cultures with wtTIM-4 expressing T cells or without drug (FIG. 34C). Normalization was relative to co-cultures for 0 days, 0 hours and 0 minutes. A2780 cells express mCherry. For FIGS. 34A and 34B, the effector to target (E: T) ratio was 1:2. For FIG. 34C, the E:T ratio is 1:1. Nilaparib=0.5 μm, olarib=1.5 μm.

CER1234 or CER 1236T cells are activated and produce IFN- γ in response to co-culture with PARP inhibitor treated a2780 ovarian cancer cells. TNF- α (FIG. 35A), IFN- γ (FIG. 35B), granzyme B (FIG. 35C) was induced by CER1234 or CER 1236T cells, but not by non-transduced cells or wtTIM-4T cells. TNF- α, IFN- γ and granzyme B were induced in the presence of PARP inhibitors but not by vehicle. Effector to target ratio of 1:1; nilapatinib. Nilaparib=0.5 μm, olarib=1.5 μm.

CER1236T cells exhibit antigen presenting cell-like functions in an in vitro system. In the E7 HLA2 restricted TCR system, CER1236-T cells support greater activation of autologous E7TCR T cells when compared to non-transduced T cells or anti-CD 19 CAR T cells, suggesting that CER-1236T cells enhance E7 antigen presentation (fig. 36A). CER1236T cell-induced E7TCR activation was dependent on antigen presentation via HLA class I system (fig. 36B). CER1236T cells can acquire antigen, a function that is considered to be a barrier to its ability to process and present antigen to the naive T cells. Engineered Antigen Presenting Cell (APC) functions of CER1236T cells may lead to initiation of a secondary immune response in vivo.

Example 8: in vitro study of CER 1236T cells in combination with octreotide

The combination of CER 1236T cells with the EGFR tyrosine kinase inhibitor, octtinib, was tested on a non-small cell lung cancer (NSCLC) cell line. Ten thousand NSCLC H1975 cells were incubated with 100nM of octtinib or vehicle (DMSO) for 24 hours and then co-cultured with 2500 CER 1236T cells at an effector to target cell ratio of 0.25:1 in the presence or absence of 4.88nM of octtinib or vehicle. Tumor cell growth was monitored in the Incucyte assay for 120 hours. As shown in fig. 39, the combination of octenib and CER 1236T cells synergized in vitro to suppress H1975NSCLC cells.

Cytokine production was also measured in CER1236T cells co-cultured with H1975 NSCLC cells pre-treated with octenib or vehicle. Ten thousand NSCLC H1975 cells were incubated with 100nM of Ornitinib or vehicle (DMSO) for 24 hours, then co-incubated with CER T cells for 96 hours at 1:4 E:T in the presence or absence of 4.88nM of Ornitinib or vehicle. Effector to target ratio was 1:4 and H1975 cells were seeded at 10,000 cells/well. Cytokine production was determined by the Ella assay. As shown in fig. 40, increased cytokine production (granzyme B, TNF α, IL-6 and ifnγ) was observed for CER1236T cells + octtinib compared to CER1236T cells + vehicle and non-transduced T cells with or without octtinib.

Proliferation of CER1236T cells co-cultured with H1975 NSCLC cells pretreated with octenib or vehicle was also measured. NSCLC H1975 cells were pre-treated with 100nM of octenib or DMSO for 24 hours, followed by successively lower doses of 4.88nM of octenib/DMSO in the presence of non-transduced T cells or CER-1236T cells at an effector to target ratio of 1:4. The fold expansion of total cd3+ T cells was determined by quantitative flow cytometry using quantitative beads over a period of time (48 hours, 96 hours, 120 hours, and 144 hours). Experiments were performed twice, with 5,000 target H1975 cells at a time, and 10,000 target H1975 cells at a time. As shown in fig. 41, octenib promoted CER1236T cell proliferation compared to CER1236T cell + vehicle and non-transduced T cells with or without octenib.

FIG. 42 shows expansion of CD3+ T cells and reduction of H1975 target cells in experiments with CER 1236T cells co-cultured with H1975NSCLC cells (4:1 effector: target ratio) pre-treated with octenib or vehicle for 120 hours as measured by flow cytometry. As shown in the bottom row of fig. 42, the cd3+ T cells proliferated the highest and the H1975 cells were the lowest in the CER 1236T cell samples co-cultured with H1975 cells treated with octenib.

Example 9: comparison of CER 1234T cells and CER 1236T cells

T cells obtained from three different donors were transduced with CER1234 or CER1236 and incubated on titrating doses of plate-bound phosphatidylserine. As shown in fig. 44, plate-bound phosphatidylserine can induce ifnγ in all donor T cells transduced with CER1236, and this induced response appears to be more consistent in CER 1236T cells than in CER1234T cells. Figure 45 shows ifnγ production in CER1234T cells or CER 1236T cells by donors in response to increasing doses of plate-bound phosphatidylserine. CER 1236T cells from all donors showed strong activation. For donors 32 and 38, CER1234T cells showed lower activation, but CER1234T cells from donor 31 did not pass. Figure 46 shows ifnγ production in CER1234T cells or CER 1236T cells by the donor in response to increasing doses of plate-bound phosphatidylserine on day 14 after thawing. Likewise, CER 1236T cells produced more ifnγ in all donors than CER1234T cells.

Fig. 47 shows T cell expansion in CER 1236T cells in response to increasing doses of plate-bound phosphatidylserine (left), ifnγ production (center), and cell viability (right). 24-hour ifnγ stimulation by plate-bound phosphatidylserine was consistent with day 5 expansion after CER 1236T cell activation. Fig. 48 shows T cell expansion (left), ifnγ production (center), and cell viability (right) in CER 1234T cells in response to increasing doses of plate-bound phosphatidylserine. 24-hour ifnγ stimulation by plate-bound phosphatidylserine was consistent with day 5 expansion after CER 1234T cell activation.

After 48 hours of culture alone or co-culture with phosphatidylserine positive HPV E7 oncoprotein positive cells (TMEM 30A-/-SCC 152), the Antigen Presenting Cell (APC) like function of the non-transduced T cells or CER1183, CER1161, CER1234 or CER 1236T cells (fig. 49 is a schematic of the construct) was assessed, followed by positive selection of T cells and co-culture with CELL TRACE violet labeled HPV E7 TCR T cells for a period of 4-6 days. Flow cytometry was used in repeated experiments to analyze activation markers HLA-DR and CD25 on E7 TCR T cells (FIGS. 50-54).

In FIG. 50, a significant difference in HLA-DR MFI was observed between non-transduced T cells and CER 1236T cells. No significant differences were observed between CER1234 and CER 1236T cells. In fig. 51, a significant difference in HLA-DR MFI was observed between non-transduced T cells and CER 1236T cells, and CER1236 appeared to have better antigen presenting function than CER1234 in two different experiments. In fig. 52, both CER1234 and CER 1236T cells showed better antigen presenting function than non-transduced T cells. No significant difference was observed between CER1234 and CER 1236. In fig. 53, both CER1234 and CER 1236T cells showed better antigen presenting function than non-transduced T cells. No significant difference was observed between CER1234 and CER 1236. In FIG. 54, a significant difference in HLA-DR MFI was observed between non-transduced T cells and CER 1236T cells. CER 1236T cells also showed improved antigen presenting function compared to CER1234T cells as measured by CD25 and HLA-DR.

Overall, these results indicate that CER 1236T cells consistently have better antigen presenting function than non-transduced T cells or T cells with constructs that do not bind phosphatidylserine. In some experiments, CER1234 did not exhibit any antigen presenting function on non-transduced T cells. In contrast to CER 1234T cells, CER 1236T cells consistently exhibit antigen presenting functions.

As previously described in example 5, CER-1236T cells exhibited repeated IFN- γ responses after 3 consecutive rounds of exposure to phosphatidylserine (fig. 19C). The ability of CER-1236T cells to become reactivated upon in vitro exposure to additional antigen suggests a durable effect in vivo.

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The various embodiments described above may be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in this specification and/or listed in the application data sheet, including U.S. provisional patent application No. 63/226,643 filed on month 7 and 28 of 2021, U.S. provisional patent application No. 63/226,712 filed on month 7 and 28 of 2021, U.S. provisional patent application No. 63/226,736 filed on month 7 and 28 of 2021, U.S. provisional patent application No. 63/311,016 filed on month 2 and 16 of 2022, U.S. provisional patent application number 63/311,042 filed on month 2 of 2022, U.S. provisional patent application number 63/311,043 filed on month 16 of 2022, U.S. provisional patent application number 63/311,045 filed on month 2 of 2022, U.S. provisional patent application number 63/336,972 filed on month 29 of 2022, U.S. provisional patent application number 63/336,980 filed on month 29 of 2022, U.S. provisional patent application number 63/341,999 filed on month 5 of 2022, U.S. provisional patent application number 63/342,031 filed on month 13 of 2022, international patent application number PCT/US2021/046043 filed on month 8 of 2021, international patent application number PCT/US2021/046014 filed on month 13 of 2021 and international patent application number PCT/US2021/046041 filed on month 13 of 2021. Aspects of the embodiments can be modified if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure.

Claims

1. A chimeric T cell immunoglobulin and mucin 4 (Tim 4) receptor comprising a single chain chimeric protein comprising:

(a) An extracellular domain comprising a Tim4 binding domain, said Tim4 binding domain comprising the amino acid sequence of SEQ ID No. 6:

(b) An intracellular signaling domain, wherein the intracellular signaling domain comprises:

(i) A primary CD28 intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 12, a secondary TLR2 TIR intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 17, and a tertiary CD3 zeta intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 14; or alternatively

(Ii) A primary CD28 intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 12, a secondary cd3ζ intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 14, and a tertiary TLR2 TIR intracellular signaling domain comprising the amino acid sequence of SEQ ID No. 17; and

(C) A CD28 transmembrane domain comprising the amino acid sequence of SEQ ID No. 11, which is located between and connects said extracellular domain and said CD28 intracellular signaling domain.

2. The chimeric Tim4 receptor of claim 1, wherein the chimeric Tim4 receptor further comprises a signal peptide at the N-terminus, optionally wherein the signal peptide is a Tim4 signal peptide, further optionally wherein the Tim4 signal peptide comprises the amino acid sequence of SEQ ID No. 7.

3. The chimeric Tim4 receptor according to claim 1 or 2, wherein the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID No. 18 or SEQ ID No. 18 lacking amino acids 1-24.

4. The chimeric Tim4 receptor according to claim 1 or 2, wherein the chimeric Tim4 receptor comprises the amino acid sequence of SEQ ID No. 19 or SEQ ID No. 19 lacking amino acids 1-24.

5. A polynucleotide encoding the chimeric Tim4 receptor of any one of claims 1 to 4.

6. An expression vector comprising the polynucleotide of claim 5.

7. The expression vector of claim 6, wherein the expression vector is a viral vector, wherein optionally the viral vector is a lentiviral vector.

8. An engineered cell comprising the chimeric Tim4 receptor of any one of claims 1 to 4, the polynucleotide of claim 5, or the expression vector of claim 6 or 7.

9. The engineered cell of claim 8, wherein the cell is an immune cell.

10. The engineered cell of claim 9, wherein the cell is a T cell.

11. The engineered cell of claim 10, wherein the cell is a cd4+ T cell, a cd8+ T cell, or a cd4+/cd8+ T cell.

12. The engineered cell of any one of claims 9 to 11, wherein the cell is a human cell.

13. A composition comprising the chimeric Tim4 receptor of any one of claims 1 to 4, the polynucleotide of claim 5, the vector of claim 6 or 7, or the engineered cell of any one of claims 8 to 12.

14. The composition of claim 13, further comprising a pharmaceutically acceptable excipient.

15. A method of treating a disease in a subject comprising administering the chimeric Tim4 receptor of any one of claims 1 to 4, the polynucleotide of claim 5, the vector of claim 6 or 7, or the engineered cell of any one of claims 8 to 12, or the composition of claim 13 or 14.

16. The method of claim 15, wherein the disease is cancer.

17. The method of claim 16, wherein the cancer is breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, or lung cancer; adenocarcinomas of the breast, prostate and colon; all forms of lung bronchogenic carcinoma; myeloid leukemia; melanoma; liver cancer; neuroblastoma; papillomas; amine precursor uptake and decarboxylation of the cell tumor; a vaginosis tumor; gill tumor; malignant carcinoid syndrome; carcinoid heart disease; and cancers (e.g., walker cancer, basal cell carcinoma, basal squamous cell carcinoma, brown-Pearce cancer, ductal carcinoma, ehrlich tumor, krebs 2 cancer, merkel cell carcinoma, mucinous cancer, non-small cell lung cancer, oat cell carcinoma, papillary carcinoma, hard carcinoma, bronchiolar carcinoma, squamous cell carcinoma, and transitional cell carcinoma); tissue cell disorders; malignant tissue cytopathy; leukemia; hodgkin's disease; immunoproliferative small bowel disease; non-hodgkin's lymphoma; plasmacytoma; multiple myeloma; chronic Myelogenous Leukemia (CML); plasmacytoma; reticuloendothelial tissue proliferation; melanoma; chondroblastoma; cartilage tumor; chondrosarcoma; fibroids; fibrosarcoma; giant cell tumor; histiocytoma; a fatty tumor; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngeal pipe tumor; a vegetative cell tumor; hamartoma; a stromal tumor; mesonephroma; myosarcoma; enameloblastoma; cementoma; dental tumor; teratoma; thymoma; a trophoblastic tumor; adenoma; gall bladder tumor; cholesteatoma; cylindrical tumors; cystic adenocarcinoma; cystic adenoma; granulosa cell tumors; ampholytic embryonal cytoma; liver cancer; sweat gland tumor; islet cell tumor; leydig cell tumor; papillomas; support cell tumor; follicular membrane cytoma; smooth myoma; leiomyosarcoma; myoblasts; myomas; myosarcoma; rhabdomyomas; rhabdomyosarcoma; ventricular tube membranoma; gangliocytoma; glioma; medulloblastoma; meningioma; a schwannoma; neuroblastoma; neuroepithelial tumors; neurofibromatosis; neuroma; paraganglioma; non-chromaphilic paragangliomas; vascular keratoma; vascular lymphoid hyperplasia is accompanied by eosinophilia; sclerosing hemangioma; hemangiomatosis; glomeroclavicular tumor; vascular endothelial tumors; hemangioma; vascular endothelial cell tumor; hemangiosarcoma; lymphangioma; lymphangiomyomas; lymphangiosarcoma; pineal tumor; carcinoma sarcoma; chondrosarcoma; she Zhuangnang sarcoma; fibrosarcoma; hemangiosarcoma; leiomyosarcoma; leukemia sarcoma; liposarcoma; lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasms; neurofibromatosis; cervical dysplasia and peritoneal cancer; b cell carcinomas, including B cell lymphomas (e.g., various forms of hodgkin's disease, non-hodgkin's lymphoma (NHL) or central nervous system lymphoma), leukemias (e.g., acute Lymphoblastic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), hairy cell leukemia, B-cell transformation of chronic myelogenous leukemia) and myelomas (e.g., multiple myeloma); small lymphocytic lymphoma, small lymphocytic leukemia, fahrenheit macroglobulinemia, B-cell pre-lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, bone solitary plasmacytomer, extraosseous plasmacytomer, extranodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, burkitt's lymphoma/leukemia, B-cell proliferation of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorders.

18. The method of claim 17, wherein the cancer is a B cell cancer, optionally wherein the B cell cancer is a B cell lymphoma, further optionally wherein the B cell lymphoma is a mantle cell lymphoma.

19. The method of any one of claims 15 to 18, further comprising administering an additional therapeutic agent.

20. The method of claim 19, wherein the additional therapeutic agent comprises radiation therapy, cellular immunotherapy, antibodies, immune checkpoint molecule inhibitors, chemotherapy, hormonal therapy, peptides, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, UV light therapy, electrical pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, small molecule therapy, or any combination thereof.

21. The method of claim 19 or 20, wherein the cellular immunotherapy is a chimeric antigen receptor.

22. The method of claim 19 or 20, wherein the additional therapeutic agent comprises an angiogenesis inhibitor (e.g., VEGF pathway inhibitor), a tyrosine kinase inhibitor (e.g., EGF pathway inhibitor), a receptor tyrosine kinase inhibitor, a growth factor inhibitor, a GTPase inhibitor, a serine/threonine kinase inhibitor, a transcription factor inhibitor, a B-Raf inhibitor, a MEK inhibitor, an mTOR inhibitor, an EGFR inhibitor, an ALK inhibitor, a PARP inhibitor, a ROS1 inhibitor, a BCL-2 inhibitor, a PI3K inhibitor, a VEGFR inhibitor, a BCR-ABL inhibitor, a MET inhibitor, a MYC inhibitor, an ABL inhibitor, a HER2 inhibitor, a BTK inhibitor, a H-RAS inhibitor, a K-RAS inhibitor, a PDGFR inhibitor, a TRK inhibitor, a c-KIT inhibitor, a CDK4/6 inhibitor, a FAK inhibitor, an FGFR inhibitor, an FLT3 inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, a PDGFRA inhibitor, or a RET inhibitor.

23. The method of claim 22, wherein the inhibitor is a BTK inhibitor.

24. The method of claim 23, wherein the BTK inhibitor is ibrutinib (ibrutinib), pirbutitinib (pirtobrutinib) (Loxo-305), tiratinib (tirabrutinib), tolutinib (tolebrutinib), erlotinib (evobrutinib), februtinib (fenebrutinib) (GDC-0853), acartinib (acalabrutinib), ONO-4059, capetinib (spebrutinib), zebutitinib (zanubrutinib) (BGB-3111), HM71224, or M7583.

25. The method of claim 23 or 24, wherein the cancer is mantle cell lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, small lymphocytic leukemia, fahrenheit macroglobulinemia, and marginal zone lymphoma.

26. The method of claim 22, wherein the inhibitor is an EGFR inhibitor.

27. The method of claim 26, wherein the EGFR inhibitor is octreotide (osimertinib), erlotinib, gefitinib (gefitnib), afatinib (afatinib), or dacatinib (dacomitinib).

28. The method of claim 26 or 27, wherein the cancer is non-small cell lung cancer.

29. The method of claim 22, wherein the inhibitor is a poly ADP-ribose polymerase (PARP) inhibitor.

30. The method of claim 29, wherein the PARP inhibitor is tazopanib (talazoparib), nilaparib (niraparib), lu Kapa ni (rucaparib), olaparib (olaparib), veliparib (veliparib), CEP 9722, E7016, AG014699, MK4827, BMN-673, or pamiparib (pamiparib).

31. The method of claim 29 or 30, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer, or melanoma.

32. The method of claim 31, wherein the breast cancer is a triple negative breast cancer.

33. The method of claim 31, wherein the ovarian cancer is advanced ovarian cancer.

34. The method of claim 31, wherein the prostate cancer is advanced prostate cancer.

35. The method of claim 31, wherein the lung cancer is non-small cell lung cancer.

36. The method of any one of claims 29 to 35, wherein the cancer is a breast cancer gene (BRCA) mutated cancer.

37. The method of claim 36, wherein the cancer is BRCA1 mutated cancer, BRCA2 mutated cancer, or both.

38. The method of claim 21, wherein the chimeric antigen receptor is an anti-CD 72 chimeric antigen receptor.

39. The method of claim 38, wherein the anti-CD 72 chimeric antigen receptor has a binding domain comprising heavy chain complementarity determining region 1 (HCDR 1), heavy chain complementarity determining region 2 (HCDR 2), heavy chain complementarity determining region 3 (HCDR 3), light chain complementarity determining region 1 (LCDR 1), light chain complementarity determining region 2 (LCDR 2), and light chain complementarity determining region 3 (LCDR 3) provided in table 3.

40. The method of claim 38 or 39, wherein the anti-CD 72 chimeric antigen receptor has a binding domain comprising the amino acid sequence of any one of SEQ ID NOs 131-164.

41. The method of any one of claims 38-40, wherein the anti-CD 72 chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs 182-234.

42. The method of any one of claims 38 to 41, wherein the cancer is a B cell malignancy.

43. The method of claim 42, wherein the B cell malignancy is selected from the group consisting of: b-cell Chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), acute myelogenous leukemia, chronic Myelogenous Leukemia (CML), promyelocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia, non-hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, splenic marginal zone lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, and hodgkin's lymphoma.

44. The method of claim 42 or 43, wherein the subject has a refractory B cell malignancy.

45. The method of any one of claims 42 to 44, wherein the subject was previously administered a chimeric antigen receptor that targets CD19 or CD 22.

46. The method of any one of claims 15-45, wherein the additional therapeutic agent is administered in a sub-therapeutic dose.

47. The method of any one of claims 15 to 46, wherein the additional therapeutic agent is administered to the subject sequentially or simultaneously with the chimeric Tim4 receptor.

48. A method of enhancing effector response or anti-tumor efficacy in a subject comprising administering the chimeric Tim4 receptor of any one of claims 1 to 4, the polynucleotide of claim 5, the expression vector of claim 6 or 7, or the engineered cell of any one of claims 8 to 12, or the composition of claim 13 or 14.

49. A method for enhancing the expression of ccr7+ T cells in a subject having cancer, the method comprising administering to the subject the chimeric Tim4 receptor of any one of claims 1 to 4, the polynucleotide of claim 5, the expression vector of claim 6 or 7, or the engineered cell of any one of claims 8 to 12, or the composition of claim 13 or 14; and optionally a poly ADP-ribose polymerase (PARP) inhibitor.

50. The method of claim 49, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer, or melanoma.

51. The method of claim 50, wherein the breast cancer is a triple negative breast cancer.

52. The method of claim 50, wherein the ovarian cancer is advanced ovarian cancer.

53. The method of claim 50, wherein the prostate cancer is advanced prostate cancer.

54. The method of claim 50, wherein the lung cancer is non-small cell lung cancer.

55. The method of any one of claims 49-54, wherein the cancer is a breast cancer gene (BRCA) mutant cancer.

56. The method of claim 55, wherein the cancer is BRCA1 mutated cancer, BRCA2 mutated cancer, or both.

57. The method of any one of claims 49-56, wherein the PARP inhibitor is talazolepaline, nilaparib, lu Kapa n, olaparib, veliparib, CEP 9722, E7016, AG014699, MK4827, BMN-673, pamipril, or a combination thereof.

58. The method of any one of claims 49-57, further comprising administering an additional therapeutic agent.

59. The method of claim 58, wherein the additional therapeutic agent comprises radiation therapy, cellular immunotherapy, antibodies, immune checkpoint molecule inhibitors, chemotherapy, hormonal therapy, peptides, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, UV light therapy, electrical pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, small molecule therapy, or a combination thereof.

60. The method of claim 59, wherein the cellular immunotherapy is a chimeric antigen receptor or a T cell receptor.

61. The method of claim 58 or 59, wherein the additional therapeutic agent comprises an angiogenesis inhibitor (e.g., VEGF pathway inhibitor), a tyrosine kinase inhibitor (e.g., EGF pathway inhibitor), a receptor tyrosine kinase inhibitor, a growth factor inhibitor, a GTPase inhibitor, a serine/threonine kinase inhibitor, a transcription factor inhibitor, a B-Raf inhibitor, a MEK inhibitor, an mTOR inhibitor, an EGFR inhibitor, an ALK inhibitor, a ROS1 inhibitor, a BCL-2 inhibitor, a PI3K inhibitor, a VEGFR inhibitor, a BCR-ABL inhibitor, a MET inhibitor, a MYC inhibitor, an ABL inhibitor, a HER2 inhibitor, a BTK inhibitor, an H-RAS inhibitor, a K-RAS inhibitor, a PDGFR inhibitor, a TRK inhibitor, a c-KIT inhibitor, a c-MET inhibitor, a CDK4/6 inhibitor, a FAK inhibitor, an FGFR inhibitor, an FLT3 inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, a PDGFRA inhibitor, or a RET inhibitor.

62. The method of any one of claims 49 to 61, wherein the enhanced ccr7+ expressing T cells express the chimeric Tim4 receptor.

63. A method for enhancing CD4/CD 8T cell ratio in a subject having cancer, the method comprising administering to the subject the chimeric Tim4 receptor of any one of claims 1 to 4, the polynucleotide of claim 5, the expression vector of claim 6 or 7, or the engineered cell of any one of claims 8 to 12, or the composition of claim 13 or 14; and optionally a poly ADP-ribose polymerase (PARP) inhibitor.

64. The method of claim 63, wherein the cancer is breast cancer, ovarian cancer, colorectal cancer, fallopian tube cancer, peritoneal cancer, prostate cancer, lung cancer, or melanoma.

65. The method of claim 64, wherein the breast cancer is a triple negative breast cancer.

66. The method of claim 64, wherein the ovarian cancer is advanced ovarian cancer.

67. The method of claim 64, wherein the prostate cancer is advanced prostate cancer.

68. The method of claim 64, wherein the lung cancer is non-small cell lung cancer.

69. The method of any one of claims 63-68, wherein the cancer is a breast cancer gene (BRCA) mutant cancer.

70. The method of claim 55, wherein the cancer is BRCA1 mutated cancer, BRCA2 mutated cancer, or both.

71. The method of any one of claims 63-70, wherein the PARP inhibitor is talazofamid, nilaparib, lu Kapa n, olaparib, veliparib, CEP 9722, E7016, AG014699, MK4827, BMN-673, pamipril, or a combination thereof.

72. The method of any one of claims 63-71, further comprising administering an additional therapeutic agent.

73. The method of claim 72, wherein the additional therapeutic agent comprises radiation therapy, cellular immunotherapy, antibodies, immune checkpoint molecule inhibitors, chemotherapy, hormonal therapy, peptides, antibiotics, antiviral agents, antifungal agents, anti-inflammatory agents, UV light therapy, electrical pulse therapy, high intensity focused ultrasound therapy, oncolytic virus therapy, small molecule therapy, or a combination thereof.

74. The method of claim 73, wherein the cellular immunotherapy is a chimeric antigen receptor or a T cell receptor.

75. The method of claim 72 or 73, wherein the additional therapeutic agent comprises an angiogenesis inhibitor (e.g., VEGF pathway inhibitor), a tyrosine kinase inhibitor (e.g., EGF pathway inhibitor), a receptor tyrosine kinase inhibitor, a growth factor inhibitor, a GTPase inhibitor, a serine/threonine kinase inhibitor, a transcription factor inhibitor, a B-Raf inhibitor, a MEK inhibitor, an mTOR inhibitor, an EGFR inhibitor, an ALK inhibitor, a ROS1 inhibitor, a BCL-2 inhibitor, a PI3K inhibitor, a VEGFR inhibitor, a BCR-ABL inhibitor, a MET inhibitor, a MYC inhibitor, an ABL inhibitor, a HER2 inhibitor, a BTK inhibitor, an H-RAS inhibitor, a K-RAS inhibitor, a PDGFR inhibitor, a TRK inhibitor, a c-KIT inhibitor, a c-MET inhibitor, a CDK4/6 inhibitor, a FAK inhibitor, an FGFR inhibitor, an FLT3 inhibitor, an IDH1 inhibitor, an IDH2 inhibitor, a PDGFRA inhibitor, or a RET inhibitor.

76. The method of any one of claims 63-75, wherein the enhanced CD 4T cells express the chimeric Tim4 receptor.