WO2023214212A2 - Compositions and uses of cd38 and icam1 antibodies - Google Patents

Compositions and uses of cd38 and icam1 antibodies Download PDF

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Publication number
WO2023214212A2
WO2023214212A2 PCT/IB2023/000244 IB2023000244W WO2023214212A2 WO 2023214212 A2 WO2023214212 A2 WO 2023214212A2 IB 2023000244 W IB2023000244 W IB 2023000244W WO 2023214212 A2 WO2023214212 A2 WO 2023214212A2
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seq
cysteine
isolated recombinant
recombinant antibody
residues
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PCT/IB2023/000244
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French (fr)
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WO2023214212A3 (en
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Xiaocheng Chen
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Virtuoso Binco, Inc.
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Publication of WO2023214212A3 publication Critical patent/WO2023214212A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • isolated recombinant antibodies that target cluster of differentiation (CD38) and intracellular adhesion molecule 1 (ICAM1) that comprise: a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3, and wherein the isolated recombinant antibodies have at least one of the following characteristics: (a) at least one disulfide bond formed by a pair of cysteine residues; (b) a far UV circular dichroism peak at a wavelength between 195 nm and 210 nm when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH
  • the LC comprises at least 85%sequence identity to SEQ ID NO: 1. In some embodiments the LC comprises at least 90%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2.
  • the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3.
  • the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  • the isolated recombinant antibody comprises at least two of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least three of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least four of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least five of the characteristics. In some embodiments, an isolated recombinant antibody comprises at least one disulfide bond. In some embodiments, the at least one disulfide bond is an intrachain disulfide bond. In some embodiments, the at least one disulfide bond is an interchain disulfide bond. In some embodiments, the interchain disulfide bond is between the HC-Knob and the HC-Hole.
  • the at least two disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least three disulfide bonds are formed by pairs of cysteine residues. In some embodiments, least four disulfide bonds formed by pairs of cysteine residues. In some embodiments, at least five disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least six disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least seven disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least eight disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least nine disulfide bonds are formed by pairs of cysteine residues.
  • At least ten disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least eleven disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least twelve disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least thirteen disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least fourteen disulfide bonds are formed by pairs of cysteine residues. In some embodiments, the pair of cysteine residues comprises Cysteine 23 and Cysteine 88 of SEQ ID NO: 1 In some embodiments, the pair of cysteine residues comprises Cysteine 133 and Cysteine 197 of SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1.
  • the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ tD NO: 2. In some embodiments, the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3.
  • the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO:2 and Cysteine 263 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3.
  • the pair of cysteine residues comprises Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  • the at least one disulfide bond formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: I; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO:
  • the at least seven disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
  • the at least ten disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQEQEQ ID NO
  • the at least 12 disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
  • the at least 13 disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: i and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQEQ ID NO:
  • the 14 disulfide bonds are formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cystein
  • an isolated recombinant antibody comprises a far UV circular dichroism peak at a wavelength between 200 nm and 205 nm. In some embodiments, an isolated recombinant antibody comprises a near UV circular dichroism peak at a wavelength between 270 nm and 290 nm. In some embodiments, the near UV circular dichroism peak is at a wavelength between 280 nm and 290 nm. In some embodiments, an isolated recombinant antibody comprises a T 0 between 59 °C to 61 °C, Tm 1 is between 69 °C to 71 °C, and Tm 2 is between 71 °C to 74 °C.
  • an isolated recombinant antibody comprises at least one N-glycan moiety comprising N-acetylglucosamine (GlcNAc) , hexose, fucose, N-Acetylneuraminic acid (Neu5Ac) , or N-Glycolylneuraminic acid (Neu5Gc) .
  • the at least one N-gtycan moiety comprises GlcNAc, hexose, or Neu5Gc.
  • the at least one N-glycan moiety comprises GlcNAc and hexose. In some embodiments, the at least one N-glycan moiety comprises GlcNAc, hexose, and Neu5Gc. In some embodiments, the at least one N-glycan moiety comprises at least two GlcNAc moieties and at least two hexose moieties. In some embodiments, the at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties.
  • the at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises five GleNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAe moieties and four hexose moieties.
  • the at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glyean moiety comprises five GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, and one Neu5Ae moiety.
  • the at least one N-glycan moiety comprises two GlcNAc moieties and eight hexose moieties. In some embodiments, the at least one N-glycan moiety is at Asparagine 300 of SEQ ID NO: 2. In some embodiments, the at least one N-glycan moiety is at Asparagine 334 of SEQ ID NO: 3.
  • the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1.
  • the isolated recombinant antibodies comprise a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2.
  • the heavy chain hole (HC-Hole) amino acid sequence has at least 80%sequence identity to SEQ ID NO: 3.
  • a plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
  • the plurality of isolated recombinant antibodies comprise greater than 95%monomer. In some embodiments, the plurality of isolated recombinant antibodies, comprises greater than 99%monomer. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of greater than or equal to 1.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 2.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 5.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 10.0 mg/mL.
  • the plurality of isolated recombinant antibodies comprise a concentration of at least 15.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 20.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a buffered solution having a pH less than or equal to 5.5. In some embodiments, the buffered solution comprises one or more of acetate, phosphate, or histidine. In some embodiments, the buffered solution comprises acetate at a concentration greater than 15mM. In some embodiments, the buffered solution comprises sucrose.
  • the plurality of isolated recombinant antibodies comprise sucrose at a concentration greater than or equal to 5%w/v.
  • the buffered solution comprises polysorbate 80.
  • polysorbate 80 is at a concentration greater than or equal to 0.01%w/v.
  • the plurality of isolated recombinant antibodies comprise greater than 90%monomer at a concentration of greater than or equal to 20.0 mg/mL in 20 mM acetate, 8%(w/v) sucrose, 0.02% (w/v) polysorbate 80, pH of 5.0.
  • the cancer comprises a cell that expresses CD38 and ICAM1.
  • the cancer comprises a solid tumor or a hematological malignancy.
  • the cancer comprises the hematological malignancy.
  • the hematological malignancy is multiple myeloma, lymphoma, or Burkitt lymphoma.
  • the cancer is a lung cancer or a prostate cancer.
  • the cancer is a solid tumor.
  • the solid tumor comprise a refractory solid tumor. In some embodiments, the solid tumor comprises a relapsed solid tumor. In some embodiments, the multiple myeloma comprises a refractory multiple myeloma. In some embodiments, the multiple myeloma comprises relapsed multiple myeioma. In some embodiments, the lymphoma comprises a refractory lymphoma. In some embodiments, the lymphoma comprises a relapsed lymphoma. In some embodiments, the subject has received a diagnosis of a relapsed or refractory solid tumor. In some embodiments, the subject has received a diagnosis of relapsed or refractory multiple myeloma. In some embodiments, the subject has received a diagnosis of relapsed or refractory lymphoma.
  • FIG. 1A, 1B, and 1C illustrate manufacturing and purification processes for an isolated recombinant antibody described herein.
  • FIG. 2 illustrates schematic representation of a 3-chain isolated recombinant antibody described herein that targets CD38 and ICAM1. Shown is a light chain (LC) , a heavy chain-knob (HC-Knob) , and a heavy chain-hole (HC-Hole) .
  • LC light chain
  • HC-Knob heavy chain-knob
  • HC-Hole heavy chain-hole
  • FIG. 3 illustrates theoretical disulfide bond linkages for an isolated recombinant antibody described herein.
  • FIG. 4 illustrates overlaid UV chromatograrns of non-reduced and reduced peptide maps of an isolated recombinant antibody described herein by Lys-C/trypsin sequential digestion with peak annotation.
  • FIG. 5 illustrates far-UV CD spectra of an isolated recombinant antibody described herein.
  • FIG. 6 illustrates near-UV CD spectra of an isolated recombinant antibody described herein.
  • FIG. 7 illustrates DSC thermogram of an isolated recombinant antibody described herein.
  • FIG. 8A illustrates a full-view chromatogram of N-glycans for an isolated recombinant antibody described herein
  • FIG. 8B illustrates a zoom-in chromatograrn of N-glycans of the recombinant antibody.
  • FIG. 9 illustrates the symbol structures of N-glycans.
  • Antibodies that bind to CD38 are useful for the treatment of cancers that express CD38.
  • Anti-CD38 antibodies are thought to kill cancer cells by various mechanisms including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.
  • One such antibody, daratumumab is approved for the treatment of adults with multiple myeloma.
  • Reduced CD38 expression can limit the efficacy of anti-CD38 antibodies.
  • Proposals to overcome this limitation include treatment with an antibody having a higher affinity for CD38, treatment with an antibody that binds to a different epitope on CD38, treatment with an antibody that more effectively inhibits CD38 enzymatic activity, treatment with a tetravalent anti-CD38 antibody, and concurrent treatment with all-trans retinoic acid to increase CD38 expression.
  • Antibody-based therapeutics have emerged as effective cancer treatment options due to the specificity and affinity of the antibody to target binding and ease of chemical and molecular modifications.
  • approved antibody-based therapy includes monoclonal antibodies such as rituximab, tositumomab, and trastuzumab; and bispecific T-cell engager such as Blinatumomab.
  • low receptor copy numbers on a target cell, or low affinity toward an antigen has hindered the therapeutic effect of an antibody-based therapy.
  • non-specificity of an antibody toward a target antigen or the presence of a target in both cancer and non-cancer cells have further limited the use of an antibody-based therapy.
  • CD38 also known as cyclic ADP ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain.
  • CD38 mediates cytokine secretion and activation and proliferation of lymphocytes (Funaro et al, J Immunology 145: 2390-6, 1990; Guse et al, Nature 398: 70-3, 1999) , and via its NAD glycohydrolase activity regulates extracellular NAD+ levels which have been implicated in modulating the regulatory T-cell compartment (Adriouch et al., 14: 1284-92, 2012; Chiarugi et al., Nature Reviews 12: 741-52, 2012) .
  • CD38 is upregulated in different types of cancer, in particular, in hematologic malignancies such as multiple myeloma.
  • ICAM1 also known as CD54, is an lg-like cell adhesion molecule.
  • ICAM1 is an endothelial-and leukocyte-associated transmembrane protein and is involved in stabilizing cell-cell interactions and facilitating leukocyte endothelial transmigration.
  • ICAM1 is expressed in various cell types, including endothelial cells and leukocytes, and can be expressed or overexpressed in different cancer cells such as myeloma, pancreatic cancer, glioma, lung cancer, melanoma, colorectal cancer, and lymphoma.
  • isolated recombinant antibodies that target CD38 and ICAM1.
  • the isolated recombinant antibodies comprise beneficial characteristics related to secondary structure, tertiary structure, melting temperature, or post-translational modifications, that enhance efficacy or improve stability of the recombinant antibodies that target CD38 or ICAM1.
  • isolated recombinant antibodies that target CD38 and ICAM1 comprising a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3, and wherein the isolated recombinant antibody has at least one of the following characteristics: (a) at least one disulfide bond formed by a pair of cysteine residues; (b) a far UV circular dichroism peak at a wavelength between 195 nm and 210 nm when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; (c) a near UV circular dichroism peak at
  • the isolated recombinant antibody comprises at least two of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least three of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least four of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least five of the characteristics.
  • the isolated recombinant antibody comprises two of the characteristics. In some embodiments, the isolated recombinant antibody comprises three of the characteristics. In some embodiments, the isolated recombinant antibody comprises four of the characteristics. In some embodiments, the isolated recombinant antibody comprises five of the characteristics.
  • the LC comprises at least 85%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 90%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2.
  • the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 90%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3.
  • the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO:3. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  • the HC-knob sequence does not comprise a C-terminal lysine 450. In some embodiments, the HC-knob sequence comprises an amidated C-terminal proline 448. In some embodiments the HC-knob sequence does not comprise a C-terminal lysine 450 and glycine 449.
  • the HC-hole sequences does not comprise a C-terminal lysine 484. In some embodiments, the HC-hole sequence comprises an amidated C-terminal proline 482. In some embodiments the HC-hole sequence does not comprise a C-terminal lysine 484 and glycine 483.
  • the HC-hole sequence comprises an oxidated methionine 289 or an oxidated methionine 465.
  • the HC-knob sequence comprises an oxidated methionine 255 or an oxidated methionine 431.
  • the HC-hole sequence comprises one or more ora deamidated asparagine 352, deamidated asparagine 421, and deamidated asparagine 465.
  • the HC-knob sequence comprises one or more of a deamidated asparagine 318, deamidated asparagine 387, and deamidated asparagine 437.
  • the HC-knob sequences comprises a succinimide formation at asparagine 318 or asparagine 387. In some embodiments, the HC-knob sequences comprises a succinimide formation at asparagine 352 or asparagine 421.
  • the at least one disulfide bond is an intrachain disulfide bond. In some embodiments, the at least one disulfide bond is an interchain disulfide bond. In some embodiments, the interchain disulfide bond is between the HC-Knob and the HC-Hole. In some embodiments, isolated recombinant antibody comprises at least two disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least three disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least four disulfide bonds formed by pairs of cysteine residues.
  • isolated recombinant antibody comprises at least five disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least six disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least seven disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least eight disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least nine disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least ten disulfide bonds formed by pairs of cysteine residues.
  • isolated recombinant antibody comprises at least eleven disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least twelve disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least thirteen disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least fourteen disulfide bonds formed by pairs of cysteine residues.
  • the pair of cysteine residues comprises Cysteine 23 and Cysteine 88 of SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 133 and Cysteine 197 of SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 2.
  • the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 3.
  • the pair of cysteine residues comprises Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  • the at least one disulfide bond formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: l and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQEQ ID NO:
  • the isolated recombinant antibody comprises at least seven disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 88 of
  • the isolated recombinant antibody comprises at least ten disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 88
  • the isolated recombinant antibody comprises at least 12 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 88 of
  • the isolated recombinant antibody comprises at least 13 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of
  • the isolated recombinant antibody comprises 14 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 2 and Cysteine
  • the far UV circular dichroism peak is at a wavelength between 200 nm and 205 nm. In some embodiments, the near UV circular dichroism peak is at a wavelength between 270 nm and 290 nm. In some embodiments, the near UV circular dichroism peak is at a wavelength between 280 nm and 290 nm.
  • T 0 is between 59 °C to 61 °C
  • Tm 1 is between 69 °C to 71 °C
  • Tm 2 is between 71 °C to 74 °C.
  • T 0 . Tm 1 , or Tm 2 is measured by differential scanning calorimetry (DSC) .
  • the recombinant three chain antibody comprises at least one N-glycan moiety. In some embodiments, the recombinant three chain antibody comprises at least two N-glycan moieties. In some embodiments the heavy chain knob sequence comprises at least one N-glycan moiety. In some embodiments the heavy chain known sequences comprises a N-glycan moiety at Asparagine 300. In some embodiments the heavy chain hole sequence comprises at least one N-glycan moiety. In some embodiments the heavy chain hole sequence comprises a N-glycan moiety at Asparagine 334.
  • the at least one N-glycan moiety comprises N-acetylglucosamine (GlcNAc) , hexose, fucose, N-Acetylneuraminic acid (Neu5Ac) , or N-Glycolylneuraminic acid (Neu5Gc) .
  • the at least one N-glycan moiety comprises GlcNAc, hexose, or Neu5Gc.
  • the at least one N-glycan moiety comprises GlcNAc and hexose.
  • the at least one N-glycan moiety comprises GlcNAc, hexose, and Neu5Gc.
  • the at least one N-glycan moiety comprises at least two GlcNAc moieties and at least two hexose moieties. In some embodiments, the at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties.
  • the at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises five GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties.
  • the at least one N-glycan moiety comprises five GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, and one Neu5Ac moiety. In some embodiments, the at least one N-glycan moiety comprises two GlcNAc moieties and eight hexose moieties.
  • formulation (s) comprising a population of antibodies or recombinant antibodies, such as comprising any one or a combination the antibodies or recombinant antibodies as described herein.
  • composition comprising one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients comprise histidine, sucrose, polysorbate-20, sodium phosphate, citrate, acetate, sodium chloride, potassium chloride, magnesium chloride, and calcium chloride.
  • a pharmaceutical composition wherein the pharmaceutical composition comprises the recombinant polypeptide (such as any described herein) , sodium phosphate monobasic monohydrate, sodium phosphate dibasic, citrate, acetate, histidine, heptahydrate, sodium chloride, potassium chloride, histidine, citrate, acetate, sucrose, polysorbate-20, polysorbate 80, magnesium chloride hexahydrate and calcium chloride dihydrate.
  • the pharmaceutical composition can have a pH less than or equal to 6.5, 6.0, 5.5, 5.0, or 4.5.
  • the pharmaceutical composition can have a pH greater than or equal to 4.5, 5.0, 5.5, 6.0 or 6.5.
  • the pharmaceutical composition can have a pH between 4.0 and 5.0, between 4.5 and 5.5, or between 5.0 and 6.0.
  • the pharmaceutical composition can comprise greater than or equal to 10mM, 11 mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, or 25mM Acetate.
  • the pharmaceutical composition can comprise less than or equal to 25mM, 24mM, 23mM, 22mM, 21 mM, 20mM, 19mM, 18mM, 17mM, 16mM, 15mM, 14mM, 13mM, 12mM, 1 lmM, or 10mM Acetate.
  • the pharmaceutical composition can comprise greater than or equal to 10mM, 1 lmM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, or 25mM Histidine.
  • the pharmaceutical composition can comprise less than or equal to 25mM, 24mM, 23mM, 22mM, 21mM, 20mM, 19mM, 18mM, 17mM, 16mM, 15mM, 14mM, 13mM, 12mM, 1 lmM, or 10mM Histidine.
  • the pharmaceutical composition can comprise greater than or equal to 5% (w/v) , 6% (w/v) , 7% (w/v) , 8% (w/v) , 9% (w/v) , or 10% (w/v) sucrose.
  • the pharmaceutical composition can comprise less than or equal to 10% (w/v) , 9% (w/v) , 8% (w/v) , 7% (w/v) , 6% (w/v) , or 5% (w/v) sucrose.
  • the pharmaceutical composition can comprise less than or equal to 0.05% (w/v) polysorbate 80, 0.04% (w/v) polysorbate 80, 0.03% (w/v) polysorbate 80, 0.02% (w/v) polysorbate 80, or 0.01%polysorbate 80.
  • the pharmaceutical composition can comprise greater than or equal to 0.01% (w/v) polysorbate 80, 0.02% (w/v) polysorbate 80, 0.03% (w/v) polysorbate 80, 0.04% (w/v) polysorbate 80, or 0.05%polysorbate 80.
  • the pharmaceutical composition comprises the recombinant polypeptide (such as any described herein) , 20 mM Acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0.
  • At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (e.g., by mole or by mass) of the antibodies of the population is monomeric. In some embodiments of the formulation, less than or equal to 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% (e.g., by mole or by mass) of the antibodies of the population is aggregated.
  • the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3 wherein the plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
  • LC light chain
  • HC-Knob heavy chain-knob
  • HC-Hole heavy chain hole amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3
  • the plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
  • the plurality comprises greater than 91%monomer. In some embodiments, the plurality comprises greater than 92%monomer. In some embodiments, the plurality comprises greater than 93%monomer. In some embodiments, the plurality comprises greater than 94%monomer. In some embodiments, the plurality comprises greater than 95%monomer. In some embodiments, the plurality comprises greater than 96%monomer. In some embodiments, the plurality comprises greater than 97%monomer. In some embodiments, the plurality comprises greater than 98%monomer. In some embodiments, the plurality comprises greater than 99%monomer.
  • the LC comprises at least 85%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 90%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2.
  • the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 90%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3.
  • the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  • the concentration of the isolated recombinant antibodies is greater than or equal to 1.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 2.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 5.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 10.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 15.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 20.0 mg/mL. In some embodiments, the plurality is in a buffered solution having a pH less than or equal to 5.5.
  • the buffered solution comprises one or more of acetate, phosphate, or histidine. In some embodiments, the buffered solution comprises acetate at a concentration greater than 15mM. In some embodiments, the buffered solution comprises sucrose. In some embodiments, the sucrose is at a concentration greater than or equal to 5%w/v. In some embodiments, the buffered solution comprises polysorbate 80. In some embodiments, the polysorbate 80 is at a concentration greater than or equal to 0.01%w/v.
  • the plurality comprises greater than 90%monomer at a concentration of greater than or equal to 20.0mg/mL in 20 mM acetate, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH of 5.0.
  • kits comprising a recombinant antibody (such as any described herein) or a composition (such as any described herein) , a container, and a label or package insert on or associated with the container.
  • the cancer comprises cancer cells that express CD38 or ICAM1.
  • the cancer cells that express CD38 or ICAM1 are lysed.
  • the antibody induces antibody-dependent cellular phagocytosis (ADCP) of the cancer cells that express CD38 or ICAM1.
  • the cancer is a hematological malignancy.
  • the cancer is B cell cancer.
  • the cancer is leukemia or lymphoma.
  • the hematological malignancy comprises a multiple myeloma.
  • the multiple myeloma comprises refractory multiple myeloma. In some embodiments, the multiple myeloma comprises relapsed multiple myeloma.
  • the cancer is lymphoma, and wherein the lymphoma is B-cell lymphoma. In some embodiments, the cancer is lymphoma, and wherein the lymphoma is T-cell lymphoma. In some embodiments, the lymphoma comprises refractory lymphoma. In some embodiments, the lymphoma comprises relapsed lymphoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor comprises a refractory solid tumor.
  • the solid tumor comprises a relapsed solid tumor.
  • the solid tumor is sarcoma, breast cancer, lung cancer, carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, colorectal cancer, endometrial cancer, esophageal cancer, prostate cancer, cervical cancer, kidney cancer, urothelial cancer, or head and neck cancer.
  • the solid tumor is lung cancer, and wherein the lung cancer is non-small cell lung cancer.
  • the solid tumor is lung cancer, and wherein the lung cancer is small cell lung cancer.
  • the solid tumor is a sarcoma.
  • the solid tumor is breast cancer.
  • the solid tumor is a carcinoma.
  • the solid tumor is ovarian cancer. In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is gastric cancer. In some embodiments, the solid tumor is colorectal cancer. In some embodiments, the solid tumor is head and neck cancer.
  • the method further comprises administering to the subject an anti-cancer agent.
  • the anti-cancer agent is a chemotherapeutic agent or a biologic agent.
  • the administering is sufficient to reduce or eliminate the cancer as compared to a comparable method lacking the administering.
  • an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle) .
  • At least one active agent in the composition is an antibody that specifically binds to CD38 and ICAM1.
  • the label or package insert indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.
  • the article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
  • the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer′ssolution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • a pharmaceutically-acceptable buffer such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer′ssolution, and dextrose solution.
  • ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 ⁇ L” means “about 5 ⁇ L” and also “5 ⁇ L. ” Generally, the term “about” includes an amount that would be expected to be within experimental error.
  • Antibodies and “immunoglobulins” are glycoproteins having the same structural characteristics. The terms are used synonymously. In some instances, the antigen specificity of the immunoglobulin is known.
  • antibody is used in the broadest sense, and covers fully assembled antibodies, antibody fragments that can bind antigen (e.g., Fab, F (ab’ ) 2, Fy, single chain antibodies (scFv) , diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and the like) , and recombinant peptides comprising the forgoing.
  • antigen e.g., Fab, F (ab’ ) 2
  • scFv single chain antibodies
  • the terms “individual (s) , ” “subject (s) , ” and “patient (s) ” mean any mammal.
  • the mammal is a human.
  • the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker) .
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker
  • percent (%) amino acid sequence identity with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the %amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program′s alignment of A and B, and where Y is the total number of amino acid residues in B.
  • the terms “individual (s) ” , “subject (s) ” and “patient (s) ” are used interchangeably herein and refer to any mammal.
  • the mammal is a human.
  • the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker) .
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker
  • Embodiment 1 An isolated recombinant antibody that targets CD38 and ICAM1 comprising a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3, and wherein the isolated recombinant antibody has at least one of the following characteristics:
  • LC light chain
  • HC-Knob heavy chain-knob
  • HC-Hole heavy chain hole amino acid sequence that has at least 80%sequence identity to SEQ ID NO:
  • Embodiment 2 The isolated recombinant antibody of embodiment 1, wherein the LC comprises at least 85%sequence identity to SEQ ID NO: 1.
  • Embodiment 3 The isolated recombinant antibody of any one of embodiments 1-2, wherein the LC comprises at least 90%sequence identity to SEQ ID NO: 1.
  • Embodiment 4 The isolated recombinant antibody of any one of embodiments 1-3, wherein the LC comprises at least 95%sequence identity to SEQ ID NO: 1.
  • Embodiment 5 The isolated recombinant antibody of any one of embodiments 1-4, wherein the LC comprises at least 99%sequence identity to SEQ ID NO: 1.
  • Embodiment 6 The isolated recombinant antibody of any one of embodiments 1-5, wherein the LC comprises the amino acid sequence according to SEQ ID NO: 1.
  • Embodiment 7 The isolated recombinant antibody of any one of embodiments 1-6, wherein the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2.
  • Embodiment 8 The isolated recombinant antibody of any one of embodiments 1-7, wherein the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2.
  • Embodiment 9 The isolated recombinant antibody of any one of embodiments 1-8, wherein the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2.
  • Embodiment 10 The isolated recombinant antibody of any one of embodiments 1-9, wherein the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2.
  • Embodiment 11 The isolated recombinant antibody of any one of embodiments 1-10, wherein the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2.
  • Embodiment 12 The isolated recombinant antibody of any one of embodiments 1-11 wherein the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3.
  • Embodiment 13 The isolated recombinant antibody of any one of embodiments 1-12, wherein the HC-Hole comprises at least 90%sequence identity to SEQ ID NO: 3.
  • Embodiment 14 The isolated recombinant antibody of any one of embodiments 1-13, wherein the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3.
  • Embodiment 15 The isolated recombinant antibody of any one of embodiments 1-14, wherein the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3.
  • Embodiment 16 The isolated recombinant antibody of any one of embodiments 1-15, wherein the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  • Embodiment 17 The isolated recombinant antibody of any one of embodiments 1-16, wherein the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  • Embodiment 18 The isolated recombinant antibody of any one of embodiments 1-17, wherein the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  • Embodiment 19 The isolated recombinant antibody of any one of embodiments 1-18, wherein the isolated recombinant antibody comprises at least two of the characteristics.
  • Embodiment 20 The isolated recombinant antibody of any one of embodiments 1-19, wherein the isolated recombinant antibody comprises at least three of the characteristics.
  • Embodiment 21 The isolated recombinant antibody of any one of embodiments 1-20, wherein the isolated recombinant antibody comprises at least four of the characteristics.
  • Embodiment 22 The isolated recombinant antibody of any one of embodiments 1-21, wherein the isolated recombinant antibody comprises at least five of the characteristics.
  • Embodiment 23 The isolated recombinant antibody of any one of embodiments 1-22, wherein the at least one disulfide bond is an intrachain disulfide bond.
  • Embodiment 24 The isolated recombinant antibody of any one of embodiments 1-23, wherein the at least one disulfide bond is an interchain disulfide bond.
  • Embodiment 25 The isolated recombinant antibody of any one of embodiments 1-24, wherein the interchain disulfide bond is between the HC-Knob and the HC-Hole.
  • Embodiment 26 The isolated recombinant antibody of any one of embodiments 1-25, wherein the isolated recombinant antibody comprises at least two disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 27 The isolated recombinant antibody of any one of embodiments 1-26, wherein the isolated recombinant antibody comprises at least three disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 28 The isolated recombinant antibody of any one of embodiments 1-27, wherein the isolated recombinant antibody comprises at least four disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 29 The isolated recombinant antibody of any one of embodiments 1-28, wherein the isolated recombinant antibody comprises at least five disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 30 The isolated recombinant antibody of any one of embodiments 1-29, wherein the isolated recombinant antibody comprises at least six disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 31 The isolated recombinant antibody of any one of embodiments 1-30, wherein the isolated recombinant antibody comprises at least seven disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 32 The isolated recombinant antibody of any one of embodiments 1-31, wherein the isolated recombinant antibody comprises at least eight disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 33 The isolated recombinant antibody of any one of embodiments 1-32, wherein the isolated recombinant antibody comprises at least nine disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 34 The isolated recombinant antibody of any one of embodiments 1-33, wherein the isolated recombinant antibody comprises at least ten disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 35 The isolated recombinant antibody of any one of embodiments 1-34, wherein the isolated recombinant antibody comprises at least eleven disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 36 The isolated recombinant antibody of any one of embodiments 1-35, wherein the isolated recombinant antibody comprises at least twelve disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 37 The isolated recombinant antibody of any one of embodiments 1-36, wherein the isolated recombinant antibody comprises at least thirteen disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 38 The isolated recombinant antibody of any one of embodiments 1-37, wherein the isolated recombinant antibody comprises at least fourteen disulfide bonds formed by pairs of cysteine residues.
  • Embodiment 39 The isolated recombinant antibody of any one of embodiments 1-38, wherein the pair of cysteine residues comprises Cysteine 23 and Cysteine 88 of SEQ ID NO: 1.
  • Embodiment 40 The isolated recombinant antibody of any one of embodiments 1-39, wherein the pair of cysteine residues comprises Cysteine 133 and Cysteine 197 of SEQ ID NO: 1.
  • Embodiment 41 The isolated recombinant antibody of any one of embodiments 1-40, wherein the pair of cysteine residues comprises Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1.
  • Embodiment 42 The isolated recombinant antibody of any one of embodiments 1-41, where1n the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2.
  • Embodiment 43 The isolated recombinant antibody of any one of embodiments 1-42, wherein the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 2.
  • Embodiment 44 The isolated recombinant antibody of any one of embodiments 1-43, wherein the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3.
  • Embodiment 45 The isolated recombinant antibody of any one of embodiments 1-44, wherein the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3.
  • Embodiment 46 The isolated recombinant antibody of any one of embodiments 1-45, wherein the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3.
  • Embodiment 47 The isolated recombinant antibody of any one of embodiments 1-46, wherein the pair of cysteine residues comprises Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 3.
  • Embodiment 48 The isolated recombinant antibody of any one of embodiments 1-47, wherein the pair of cysteine residues comprises Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 3.
  • Embodiment 49 The isolated recombinant antibody of any one of embodiments 1-48, wherein the pair of cysteine residues comprises Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3.
  • Embodiment 50 The isolated recombinant antibody of any one of embodiments 1-49, wherein the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3.
  • Embodiment 51 The isolated recombinant antibody of any one of embodiments 1-50, wherein the pair of cysteine residues comprises Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  • Embodiment 52 The isolated recombinant antibody of any one of embodiments 1-51, wherein the at least one disulfide bond formed by a pair of cysteine residues is selected from
  • Embodiment 53 The isolated recombinant antibody of any one of embodiments 1-52, wherein the isolated recombinant antibody comprises at least seven disulfide bonds formed by a pair of cysteine residues selected from
  • Embodiment 54 The isolated recombinant antibody of any one of embodiments 1-53, wherein the isolated recombinant antibody comprises at least ten disulfide bonds formed by a pair of cysteine residues selected from
  • Embodiment 55 The isolated recombinant antibody of any one of embodiments 1-54, wherein the isolated recombinant antibody comprises at least 12 disulfide bonds formed by a pair of cysteine residues selected from
  • Embodiment 56 The isolated recombinant antibody of any one of embodiments 1-55, wherein the isolated recombinant antibody comprises at least 13 disulfide bonds formed by a pair of cysteine residues selected from
  • Embodiment 57 The isolated recombinant antibody of any one of embodiments 1-56, wherein the isolated recombinant antibody comprises 14 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
  • Embodiment 58 The isolated recombinant antibody of any one of embodiments 1-57, wherein the far UV circular dichroism peak is at a wavelength between 200 nm and 205 nm.
  • Embodiment 59 The isolated recombinant antibody of any one of embodiments 1-58, wherein the near UV circular dichroism peak is at a wavelength between 270 nm and 290 nm.
  • Embodiment 60 The isolated recombinant antibody of any one of embodiments 1-59, wherein the near UV circular dichroism peak is at a wavelength between 280 nm and 290 nm.
  • Embodiment 61 The isolated recombinant antibody of any one of embodiments 1-60, wherein T 0 is between 59 °C to 61 °C, Tm 1 is between 69 °C to 71 °C, and Tm 2 is between 71 °C to 74 °C.
  • Embodiment 62 The isolated recombinant antibody of any one of embodiments 1-61, wherein the T 0 is 60.3 °C, Tm 1 is 70.0 °C, and Tm 2 is 74.1 °C.
  • Embodiment 63 The isolated recombinant antibody of any one of embodiments 1-62, wherein the at least one N-glycan moiety comprises N-acetylglucosamine (GlcNAc) , hexose, fucose, N-
  • Acetylneuraminic acid (Neu5Ac)
  • N-Glycolylneuraminic acid (Neu5Gc)
  • Embodiment 64 The isolated recombinant antibody of any one of embodiments 1-63, wherein the at least one N-glycan moiety comprises GlcNAc, hexose, or Neu5Gc.
  • Embodiment 65 The isolated recombinant antibody of any one of embodiments 1-64, wherein the at least one N-glycan moiety comprises GlcNAc and hexose.
  • Embodiment 66 The isolated recombinant antibody of any one of embodiments 1-65, wherein the at least one N-glycan moiety comprises GlcNAc, hexose, and Neu5Gc.
  • Embodiment 67 The isolated recombinant antibody of any one of embodiments 1-66, wherein the at least one N-glycan moiety comprises at least two GlcNAe moieties and at least two hexose moieties.
  • Embodiment 68 The isolated recombinant antibody of any one of embodiments 1-67, wherein the at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties.
  • Embodiment 69 The isolated recombinant antibody of any one of embodiments 1-68, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties.
  • Embodiment 70 The isolated recombinant antibody of any one of embodiments 1-69, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties.
  • Embodiment 71 The isolated recombinant antibody of any one of embodiments 1-70, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties.
  • Embodiment 72 The isolated recombinant antibody of any one of embodiments 1-71, wherein the at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties.
  • Embodiment 73 The isolated recombinant antibody of any one of embodiments 1-72, wherein the at least one N-glycan moiety comprises five GlcNAc moieties and three hexose moieties.
  • Embodiment 74 The isolated recombinant antibody of any one of embodiments 1-73, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and four hexose moieties.
  • Embodiment 75 The isolated recombinant antibody of any one of embodiments 1-74, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties.
  • Embodiment 76 The isolated recombinant antibody of any one of embodiments 1-75, wherein the at least one N-glycan moiety comprises five GlcNAc moieties and four hexose moieties.
  • Embodiment 77 The isolated recombinant antibody of any one of embodiments 1-76, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties.
  • Embodiment 78 The isolated recombinant antibody of any one of embodiments 1-77, wherein the at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, and one Neu5Ac moiety.
  • Embodiment 79 The isolated recombinant antibody of any one of embodiments 1-78, wherein the at least one N-glycan moiety comprises two GlcNAc moieties and eight hexose moieties.
  • Embodiment 80 The isolated recombinant antibody of any one of embodiments 1-79, wherein the at least one N-glycan moiety is at Asparagine 300 of SEQ ID NO: 2.
  • Embodiment 81 The isolated recombinant antibody of any one of embodiments 1-80, wherein the at least one N-glycan moiety is at Asparagine 334 of SEQ ID NO: 3.
  • Embodiment 82 A plurality of isolated recombinant antibodies that target CD38 and ICAM1 wherein the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3 wherein the plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
  • LC light chain
  • HC-Knob heavy chain-knob
  • HC-Hole heavy chain hole amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3
  • the plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
  • Embodiment 83 The plurality of isolated recombinant antibodies of embodiment 82, comprising greater than 95%monomer.
  • Embodiment 84 The plurality of isolated recombinant antibodies of embodiment 82, comprising greater than 99%monomer.
  • Embodiment 85 The plurality of isolated recombinant antibodies of any one of embodiments 82-84, wherein the concentration of the isolated recombinant antibodies is greater than or equal to 1.0 mg/mL.
  • Embodiment 86 The plurality of isolated recombinant antibodies of any one of embodiments 82-86, wherein the concentration of the isolated recombinant antibodies is at least 2.0 mg/mL.
  • Embodiment 87 The plurality of isolated recombinant antibodies of any one of embodiments 82-86, wherein the concentration of the isolated recombinant antibodies is at least 5.0 mg/mL.
  • Embodiment 88 The plurality of isolated recombinant antibodies of any one of embodiments 82-87, wherein the concentration of the isolated recombinant antibodies is at least 10.0 mg/mL.
  • Embodiment 89 The plurality of isolated recombinant antibodies of any one of embodiments 82-88 wherein the concentration of the isolated recombinant antibodies is at least 15.0 mg/mL.
  • Embodiment 90 The plurality of isolated recombinant antibodies of any one of embodiments 82-89, wherein the concentration of the isolated recombinant antibodies is at least 20.0 mg/mL.
  • Embodiment 91 The plurality of isolated recombinant antibodies of any one of embodiments 82-90, wherein the plurality is in a buffered solution having a pH less than or equal to 5.5.
  • Embodiment 92 The plurality of isolated recombinant antibodies of any one of embodiments 82-91, wherein the buffered solution comprises one or more of acetate, phosphate, or histidine.
  • Embodiment 93 The plurality of isolated recombinant antibodies of any one of embodiments 82-92 wherein the buffered solution comprises acetate at a concentration greater than 15mM.
  • Embodiment 94 The plurality of isolated recombinant antibodies of any one of embodiments 82-93, wherein the buffered solution comprises sucrose.
  • Embodiment 95 The plurality of isolated recombinant antibodies of embodiment 94, wherein the sucrose is at a concentration greater than or equal to 5%w/v.
  • Embodiment 96 The plurality of isolated recombinant antibodies of any one of embodiments 82-95, wherein the buffered solution comprises polysorbate 80.
  • Embodiment 97 The plurality of isolated recombinant antibodies of embodiment 96, wherein the polysorbate 80 is at a concentration greater than or equal to 0.01%w/v.
  • Embodiment 98 The plurality of isolated recombinant antibodies of any one of embodiments 82-97, comprising greater than 90%monomer at a concentration of greater than or equal to 20.0mg/mL in 20 mM acetate, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH of 5.0.
  • Embodiment 99 A method of treating a cancer in a subject in need thereof comprising administering to the subject the isolated recombinant antibody of any one of embodiments 1-80, or the plurality of isolated recombinant antibodies of embodiments 81-97, wherein the cancer comprises a cell that expresses CD38 and ICAM1.
  • Embodiment 100 The method of embodiment 99, wherein the cancer comprises a solid tumor or a hematological malignancy.
  • Embodiment 101 The method of embodiment 99, wherein the cancer comprises the hematological malignancy.
  • Embodiment 102 The method of embodiment 101, wherein the hematological malignancy is multiple myeloma, lymphoma, or Burkitt lymphoma.
  • Embodiment 103 The method of embodiment 99, wherein the cancer is a lung cancer or a prostate cancer.
  • Embodiment 104 The method of embodiment 100, wherein the solid tumor comprises a refractory solid tumor.
  • Embodiment 105 The method of embodiment 100, wherein the solid tumor comprises a relapsed solid tumor.
  • Embodiment 106 The method of embodiment 102, wherein the multiple myeloma comprises refractory multiple myeloma.
  • Embodiment 107 The method of embodiment 102, wherein the multiple myeloma comprises relapsed multiple myeloma.
  • Embodiment 108 The method of embodiment 102, wherein the lymphoma comprises refractory lymphoma.
  • Embodiment 109 The method of embodiment 102, wherein the lymphoma comprises relapsed lymphoma.
  • Embodiment 110 The method of embodiment 102, wherein the subject has received a diagnosis of relapsed or refractory solid tumor.
  • Embodiment 110 The method of embodiment 102, wherein the subject has received a diagnosis of relapsed or refractory multiple myeloma.
  • Embodiment 110 The method of embodiment 102, wherein the subject has received a diagnosis of relapsed or refractory lymphoma.
  • the drug manufacturing and purification process is a multi-step cell culture inoculation and expansion rounds. At each round of the cell expansion process, and prior to the next inoculum cycle, CHO cells were monitored for viability and cell density.
  • An exemplary flow diagram showing the manufacturing and purification process that can be used to develop the drug substance disclosed herein is depicted in exemplary FIG. 1.
  • an MCB vial from the cell bank was thawed, and cells were seeded in 50 ml Basal Medium 1 (Growth medium) as a starter culture and cultivated in 250 mL shake flasks at 37°C (36.5°C -37.5°C) growth parameters for two days. Following the two-day expansion, cells were monitored for viability and cell density (VCD) .
  • the cells from the starter culture in the previous step were expanded by sub-cultivation in Basal Medium 1 (growth medium) in 1 mL shake flasks for approximately 3 days until sufficient cells were generated to inoculate the next sub-cultivation. Cells were propagated at the same growth temperature parameters as before. Cells were monitored for VCD.
  • Basal Medium 1 growth medium
  • an inoculum of cells from the 2.8 L culture expansion were transferred into a 20 L Wave bioreactor containing Basal medium 2 (growth medium) and cultivated at the same growth temperature parameters for approximately 3 days. Cells were assessed for VCD.
  • the 2nd stage bioreactor cell expansion was initiated by transferring cells from the previous 1st stage bioreactor cell expansion into a 100 L bioreactor containing Growth medium. Cells were cultivated at the same growth temperature parameters for approximately 3 days. Cells were assessed for VCD.
  • the cell culture from the 2nd stage inoculum bioreactor was transferred to a 500 L production bioreactor containing growth medium supplemented with antifoam poloxamer.
  • Cells were propagated at the same growth temperature parameter of 37 °C (36.5 -37.5 °C) for either (a) 72 ⁇ 4 hours post inoculation or (b) until the VCD reached (5.00 -10.00) x 10 6 cells/mL, whichever occurred first, and which signaled to shift or drop growth temperature parameters down to 33.0 °C (32.5 -33.5 °C) .
  • the 500 L production bioreactor was operated in fed-batch mode with a total incubation time of about 14 days. For each production process, two different feeds and glucose were added aseptically to the bioreactor.
  • the first feed (Feed 1) was Cell Boost 7a Feed Medium while the second feed (Feed 2) was Cell Boost 7b Feed Medium.
  • the contents of the production bioreactor were harvested on day 14 or within 24 hours of a drop in viability below 60.0%, whichever occurred first.
  • the unprocessed bulk (UPB) was evaluated for the bioburden, which included testing for mycoplasma, adventitious viruses in vitro, retroviruses, and species-specific viruses.
  • the first step in the harvest and clarification process was to isolate cells and cell debri from the drug product by filtration.
  • the harvest was carried out by depth filtration using filters STAX PDP8 and STAX PDD1 and capacities of ⁇ 56 L/m 2 (primary) and ⁇ 112 L/m 2 (secondary) which was followed by 0.2 um filtration.
  • the filtrate was collected in an agitated tank and maintained at room temperature. The filtrate was evaluated for bioburden and endotoxin contaminants.
  • the Protein A affinity step was operated in bind and elute mode and was used to capture the product while allowing impurities such as host cell proteins (HCP) , DNA, and medium additives to flow through.
  • HCP host cell proteins
  • the processed harvest was loaded directly onto the Protein A column at a load temperature of 15 -26 °C and a loading capacity of 20 -40 g/L.
  • the column was then washed, and the bound product was eluted with a low pH buffer.
  • Product pooling was done by optical density (OD) A280 using Amsphere A3 as the resin.
  • the filtrate was evaluated for bioburden and endotoxin contaminants.
  • the low pH eluate from the Protein A step above was adjusted to pH 3.60 (3.50 -3.70) with 1M Acetic Acid and held for 60, ⁇ 120 minutes at 15 -26°C for viral inactivation and neutralization. At post-neutralization, the eluate was adjusted to pH 5.50 (5.40 -5.60) and held for 60 minutes with 1M Tris base. The eluate was then assessed for bioburden and endotoxin contaminants.
  • the pH adjusted Protein A eluate from the previous step was filtered through the PDD1 depth filter which had been equilibrated using 30 mM Sodium Acetate at a load pH 5.5 and temperature of 15 -26 °C and washed. Filtration through the PDD1 depth filter used a nominal pore size of ⁇ 0.5 ⁇ m, a load capacity of ⁇ 1196 g/m 2 and load conductivity of 2.1mS/cm (1.9 -2.5 mS/em) . The eluate was evaluated for bioburden and endotoxin contaminant. Next, the intermediate depth filter pool was then filtered through a 0.2 ⁇ m filter.
  • the AEX chromatography operation was performed using the flow-through mode where potential virus contaminants and impurities were bound to the column while the product flowed through.
  • the AEX column was equilibrated, and the intermediate depth filter pool was loaded at a temperature of 15 -26 °C, loading capacity of 80 -169 g/L, and then collected as a flow through elution pool.
  • the elution pool was washed with equilibration buffer and pooled by OD A280 using the Praesto Q65 resin.
  • the filtrate was evaluated for bioburden and endotoxin contaminants.
  • the AEX pool was next filtered through a 0.2 ⁇ m sterile filter.
  • the filtered AEX pool was further purified over a cation exchange chromatography (CEX) .
  • CEX cation exchange chromatography
  • the CEX chromatography operation was performed in bind and elute mode.
  • the column was equilibrated with a NaCl buffer at a load pH of 5.50 (5.30 -5.70) and load temperature of 15 -26 OC, and then loaded with the AEX pool.
  • the CEX column was then washed, and the product was step eluted with a NaC1 buffer at the same load condition, with pooling by OD A280 using Capto SP ImpRes as a resin.
  • the filtrate was evaluated for bioburden and endotoxin contaminants.
  • the resultant eluate was filtered through a 0.2 ⁇ m filter to remove viral contaminant.
  • the CEX pool eluted from the previous CEX procedure was adjusted for conductivity as necessary, with WFI to conductivity ⁇ 7 mS/cm and then filtered through a Viresolve Magnus 2.2 shield pre-filter, followed by a Planova 20N virus nanofilter with a filter capacity at ⁇ 968.6 g/m 2 , ⁇ 102.7 L/m 2 and operating pressure of 0.8 bar (0.7 -1.0 bar) .
  • the virus filter pool was then filtered through a 0.2 ⁇ m filter.
  • the Planova 20N nanofilter was tested for integrity prior to and after virus filtration and was additionally tested for bioburden and endotoxin contaminants.
  • the eluate from the virus filtration pool was next diluted with formulation buffer to a target protein concentration of 20.0 g/L. Polysorbate 80 was added to a final concentration of 0.02% (w/v) .
  • the concentrated eluate from the virus filtration pool was then processed through a buffer exchange into diafiltration buffer using Pellicon 3 Biomax 30kD molecular weight cut off UF/DF membranes, at a membrane capacity of ⁇ 500 g/m 2 .
  • the UF/DF pool was evaluated for bioburden, endotoxin contaminants and for protein concentration. In the next procedure, the UF/DF pool was filtered through a 0.2 ⁇ m filter.
  • the UF/DF pool was diluted to the target concentration of 20.0 g/L with diafiltration buffer. Polysorbate 80 was added to a final concentration of 0.02% (w/v) . The pool was further tested for bioburden, endotoxin contaminants and protein concentration. The formed the drug substance was then filtered and packaged as described below.
  • the formulation buffer was 20 mM Acetate, 8%sucrose, 0.02%PS80, pH 5.0.
  • the formulated drug substance from the previous steps was filtered through a 0.2 ⁇ m filter that had been tested for integrity.
  • the drug substance was dispensed or filled into 1 L PETG (polyethylene terephthalate glycol) bottles in an appropriate number of bottles according to specified or required volumes. Following sterile filtration, integrity testing was performed on the filters and the drug substance was tested for bioburden, endotoxin contaminants and for protein concentration.
  • PETG polyethylene terephthalate glycol
  • the drug molecule is a bispecific antibody (BsAb) .
  • the molecule consists of one light chains (LC) and two distinct heavy chains (HC-Knob and HC-Hole) .
  • the schematic representation of the molecule is shown in FIG. 2.
  • LC is kappa sub-type light chain
  • HC-Knob is immunoglobulin G1 (IgG1) sub-type heavy chain.
  • HC-Hole consists of a scFv (single-chain variable fragment) and CH2 (constant heavy chain 2) domain, CH3 (constant heavy chain 3) domain of IgG1 sub-type heavy chain.
  • scFv For the scFv, a scFv-VL (light chain variable region) and a scFv-VH (heavy chain variable region) are connected with a (G4S) 3 linker.
  • the “knob-into-hole” approach is adopted to tackle the problem of the homodimerization by substituting a large amino acid for a small one in the CH3 domain (the “knob” ) of HC-Knob and vice versa (the “hole” ) of HC-Hole.
  • Disulfide bond linkages are important in protein folding and play a significant role in both protein structure and functions.
  • the number of disulfide bonds and their positions are critical attributes for biopharmaceuticals to ensure safety and efficacy.
  • the drug molecule has 26 cysteine residues, as shown in FIG. 3, which are cross-linked by 3 inter-chain disulfide bonds (one set of disulfide bonds between the HC-Knob and light chains, and another two sets between the HC-Knob and HC-Hole chains) and 10 intra-chain disulfide bonds.
  • 11 disulfide bond related peptides (DS 1 to DS 11) are expected by non-reduced Lys-C/trypsin sequential digestion, because amino acid sequence 226 T-K 450 of CH2 and CH3 domain of HC-Knob are identical with sequence 260 Y-K 484 of HC-Hole.
  • Two inter-chain disulfide bonds in the hinge region between the heavy chains are observed in one peptide (DS6) upon the current digestion.
  • Non-reduced peptide mapping approach is often used to analyze disulfide bond linkages.
  • the protein samples were alkylated using N-ethylmaleimide (NEM) to protect free cysteines, then treated by Lys-C/trypsin sequential digestion.
  • NEM N-ethylmaleimide
  • the digested samples were divided into two aliquots. One aliquot was reduced with tris (2-carboxyethyl) phosphine (TCEP) (reduced sample, labeled as R) and the other was taken as control (non-reduced sample, labeled as NR) .
  • TCEP tris (2-carboxyethyl) phosphine
  • R reduced sample
  • NR non-reduced sample
  • the peptides were injected into an Agilent/UHPLC 1290 coupled with Thermo/Orbitrap Fusion mass spectrometer for data collection.
  • the peptides were separated with an Agilent/Poroshell SB-C18 column, and the UV chromatograms were acquired with UV detector at the wavelength of 214 nm. The peptides were further detected by the mass spectrometer operating with full MS scan followed by tandem mass spectrometry (MS/MS) scans.
  • MS/MS tandem mass spectrometry
  • DS1b, DS3b, DS4b/c, DS5b, DS6b/c, DS8b, DS9b and DS11b are miss-cleaved peptides for DS1a, DS3a, DS4a, DS5a, DS6a, DS8a, DS9a and DS11a, respectively (FIG. 4) .
  • some UV peak differences in the non-reduced peptide maps caused by sample preparation were confirmed and the detailed information is not shown in Table 2.
  • the UV peaks at RT 47.0 min, 51.4 min, 67.6 min, 87.3 min and 91.7 min were caused by a specific cleavages.
  • the UV peak at RT 95.4 min was caused by over-alkylation
  • the detailed masses of disulfide bond related peptides for 2368S210720YRS are listed in Table 2.
  • the peak annotation of non-reduced peptides (DS1-DS11) is shown in FIG. 4. All predicted disulfide bond related peptides were detected, and their mass differences were within 10.0 ppm compared with the theoretical ones.
  • DS lb, DS3b, DS4b/c, DS5b, DS6b/c, DS8b, DS9b and DS11b were miss-cleaved peptides for DS1a, DS3a, DS4a, DS5a, DS6a, DS8a, DS9a and DS11a, respectively.
  • HC refers to heavy chain and LC refers to light chain.
  • the RT was reported by PMI software.
  • DS1b, DS3b, DS4b/c, DS5b, DS6b/c, DS8b, DS9b and DS11b were miss-cleaved peptides for DS1a, DS3a, DS4a, DS5a, DS6a, DS8a, DS9a and DS11a, respectively. The difference was calculated by the following equation and the results were reported with 1 decimal place.
  • CD Crcular dichroism
  • a spectroscopic technique that measures the differential absorption of left and right-handed circularly polarized light in an optically active substance. It is widely used to characterize higher order structures of protein. Chiral optical properties of proteins are originated from the asymmetric centers of their amino acid components. Proteins exhibit CD signals in the far-UV and near-UV regions characteristic of protein secondary and tertiary structures, respectively.
  • the secondary structure consistency of 2368S210108Y-STD and 2368S210720YRS can be confirmed by comparing their CD spectra.
  • the CD spectra of 2368S210108Y-STD and 2368S210720YRS in the far-UV region (190-260 nm) are shown in FIG. 5.
  • the ⁇ -sheet and random coil were the main secondary structure compositions.
  • the spectral similarity/structure consistency analysis was confirmed by calculating the correlation coefficient of two spectra with “Quick Compare” tool of OPUS software. No significant difference was observed in the far-UV CD between 2368S210108Y-STD and 2368S210720YRS, revealing the consistency of the secondary structure.
  • Near-UV CD spectrum provides information on the protein tertiary structure.
  • the CD spectral pattern in the 250-350 nm region is determined by the absorption, dipole orientation and the nature of the surrounding environment of the phenylalanine (250-270 nm) , tyrosine (270-290 nm) , and tryptophan (290-305 nm) , respectively.
  • the protein samples were diluted with 20 mM Acetate, 8%sucrose, 0.02%PS80, pH 5.0 to 1.0 mg/mL.
  • the data collection and analysis were performed by Applied Photophysics Chirascan V100 CD spectrometer and Chirascan software, respectively.
  • the CD spectra of 2368S210108Y-STD and 2368S210720YRS in the near-UV region (250-350 nm) are shown in FIG. 6.
  • the spectral similarity/structure consistency analysis was confirmed by calculating the correlation coefficient of two spectra with “Quick Compare” tool of OPUS software. No significant difference was observed in the near-UV CD of 2368S210108Y-STD and 2368S210720YRS, revealing the consistency of the tertiary structure.
  • DSC Different scanning calorimetry
  • Sample 2368S210720YRS was analyzed with MicroCal DSC from Malvem.
  • the protein sample was diluted to 1 mg/mL with 20 mM Acetate, 8%sucrose, 0.02%PS80, pH 5.0 before analysis. 400 ⁇ L of the corresponding formulation buffer was added to a 96-well plate as the reference and 400 ⁇ tL of the protein sample was added.
  • the samples were heated from 10 °C to 95 °C at a heating rate of 90 °C/h in the capillary DSC system.
  • the DSC data was analyzed and fitted with MicroCal PEAQ-DSC Software 1.51. The results are shown in FIG. 7.
  • the T Onset was determined as 60.3 °C and the two transition mid-point temperatures (T m1 and T m2 ) were determined as 70.0 °C and 74.1 °C.
  • Glycosylation can play a critical role in protein stability, in vivo activity, solubility, serum half-life and immunogenicity. N-glycan analysis provides an indication on the potential safety and efficacy of therapeutic antibodies.
  • N-glycan analysis of the recombinant antibody was performed by releasing the N-glycans from glycoproteins with Rapid PNGase F. After enzymatic digestion, ice-cold ethanol was added into the solution to precipitate the deglycosylated protein. The supernatant was dried for 2-AB (2-Aminobenzamide) labeling. The labeled glycans were separated and detected with hydrophilic interaction liquid chromatography (HILIC) coupled to fluorescence detector (FLD) on UPLC system. Finally, each individual glycan was quantified by their relative peak area. The FLD chromatograms of N-glycans in the recombinant antibody are shown in FIG. 8A and FIG. 8B, and the detailed information of identified N-glycans is shown in Table 2. The major N-glycan forms of the recombinant antibody were G0 and G1 as depicted in structures in FIG. 9.
  • N-glycans follows GlcNAc-Hexose-Fucose-Neu5Ac-Neu5Gc.
  • 33000 represents GicNAc (3) -Hexose (3) -Fucose (0) -Neu5Ac (0) -Neu5Gc (0) .
  • a and b represent glycan isomers that show different retention.
  • G1a and G1b are isomers, thus are grouped to be G1.
  • the recombinant antibody was dialyzed into eleven formulation buffers at a concentration of 20.0mg/mL represented in Table 4 below. A sample of each formulation was run on HPLC to determine the precent aggregate in solution. The recombinant antibody was more stable in acetate and histidine buffer that citrate or phosphate buffer (PB) . The recombinant antibody was found to be more stable in low-pH buffers.
  • composition an isolated recombinant antibody targeting CD38 and ICAM1 (an afucosylated humanized Fc-modified immunoglobulin G1 bispecific antibody, hereinafter referred to as “composition” which is described herein) in trial participants (referred to as “subject” or “subjects” hereinafter) with relapsed or refractory multiple myeloma, lymphoma, or solid tumors.
  • Inclusion criteria a random population of 110 adult participants 18 years and older are enrolled in the trial following a histologic diagnosis of a relapsed or refractory solid tumor, refractory myeloma, or lymphoma with measurable or evaluable disease. Subjects must have progressed following all therapies of known, potential clinical benefit or for whom treatments of known clinical benefit are contraindicated. Subjects must have adequate kidney, liver, and hematologic function with an Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2. The trial is conducted in two parts:
  • Dose escalation is conducted to evaluate increasing doses of the composition to identify the maximum tolerated dose (MTD) or recommended Phase 2 dose (RP2D) .
  • MTD maximum tolerated dose
  • RP2D recommended Phase 2 dose
  • subjects or patient population with a diagnosis ora relapsed or refractory multiple myeloma, lymphoma or solid tumors, with measurable or evaluable disease enrolled in the trial receive the composition administered as an intravenous infusion weekly for 6 weeks then every 2 weeks.
  • the first subject enrolled receives the lowest dose of the composition. Once the lowest dose is shown to be safe, an additional subject is enrolled at the next higher dose.
  • Subjects continue to be enrolled into either single or multiple patient groups receiving increasing doses until the MTD or RP2D is reached.
  • Occurrence of general toxicity the incidence of treatment-emergent serious adverse events such as toxicity and change from baseline in safety parameters is monitored for a period of 30 months.
  • Occurrence of dose limiting toxicity the incidence of dose limiting toxicity is monitored during the first cycle (cycle 1) of the dose escalation for a period covering the first 21 days of administering the composition (dosing period) .
  • the serum concentrations of the composition are measured to determine the changes in serum levels from levels seen at baseline. Measurements are conducted for the duration of the trial for a period of 30 months.
  • the serum levels of the composition (antidrug) and levels of neutralizing antibodies are measured to determine the changes in levels of the antidrug and neutralizing antibodies since baseline. Measurements are conducted for the duration of the trial for a period of 30 months.
  • Measure objective response which can be assessed by International Myeloma Working Group (IMWG) for multiple myeloma, the Lugano criteria for lymphoma or RECIST 1.1 for solid tumors. Measurements are conducted for the duration of the trial for a period of 30 months.
  • IMWG International Myeloma Working Group
  • Measure time to response and duration of response which can be assessed by IMWG for multiple myeloma or the Lugano criteria for lymphoma or RECIST 1.1 for solid tumors. Measurements are conducted for the duration of the trial for a period of 30 months.
  • Measure progress-free survival which can be assessed by IMWG for multiple myeloma or the Lugano criteria for lymphoma or RECIST 1.1 for solid tumors. Measurements are conducted for the duration of the trial for a period of 30 months.
  • Measure tumor expression which can be evaluated by measuring ICAM1 and CD38 expression with clinical outcomes. Measurements are conducted for the duration of the trial for a period of 30 months.
  • HIV human immunodeficiency virus

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Abstract

Disclosed herein are recombinant CD38 and ICAM1 antibody compositions and uses of these compositions in treating cancer.

Description

COMPOSITIONS AND USES OF CD38 AND ICAM1 ANTIBODIES
CROSS REFERENCE
This application claims priority to PCT application no. PCT/IB2022/000324, filed on May 6, 2022, the entirety of which is hereby incorporated by reference herein.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 18, 2023, is named 55429-741_602_SL. xml and is 40, 348 bytes in size.
SUMMARY
Disclosed herein are isolated recombinant antibodies that target cluster of differentiation (CD38) and intracellular adhesion molecule 1 (ICAM1) that comprise: a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3, and wherein the isolated recombinant antibodies have at least one of the following characteristics: (a) at least one disulfide bond formed by a pair of cysteine residues; (b) a far UV circular dichroism peak at a wavelength between 195 nm and 210 nm when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; (c) a near UV circular dichroism peak at a wavelength between 270 nm and 300nm when the isolated recombinant 3 antibody is formulated at a concentration of 1.0 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; (d) an onset melting temperature (T0) between 58 ℃ to 62 ℃, a transition midpoint temperature 1 (Tm1) between 68 ℃ to 71 ℃, a transition midpoint temperature 2 (Tm2) between 72 ℃ to 75 ℃, when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; and (e) at least one N-glycan moiety.
In some embodiments the LC comprises at least 85%sequence identity to SEQ ID NO: 1. In some embodiments the LC comprises at least 90%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the isolated recombinant antibody comprises at least two of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least three of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least four of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least five of the characteristics. In some embodiments, an isolated recombinant antibody comprises at least one disulfide bond. In some embodiments, the at least one disulfide bond is an intrachain disulfide bond. In some embodiments, the at least one disulfide bond is an interchain disulfide bond. In some embodiments, the interchain disulfide bond is between the HC-Knob and the HC-Hole. In some embodiments, the at least two disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least three disulfide bonds are formed by pairs of cysteine residues. In some embodiments, least four disulfide bonds formed by pairs of cysteine residues. In some embodiments, at least five disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least six disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least seven disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least eight disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least nine disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least ten disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least eleven disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least twelve disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least thirteen disulfide bonds are formed by pairs of cysteine residues. In some embodiments, at least fourteen disulfide bonds are formed by pairs of cysteine residues. In some embodiments, the pair of cysteine residues comprises Cysteine 23 and Cysteine 88 of SEQ ID NO: 1 In some embodiments, the pair of cysteine residues comprises Cysteine 133 and Cysteine 197 of SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1.
In some embodiments, the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ tD NO: 2. In some embodiments, the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 232 of SEQ ID  NO:2 and Cysteine 263 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3.
In some embodiments, the pair of cysteine residues comprises Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3. In some embodiments, the at least one disulfide bond formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: I; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3. In some embodiments, the at least seven disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3. In some embodiments, the at least ten disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3  and Cysteine 462 of SEQ ID NO: 3. In some embodiments, the at least 12 disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3. In some embodiments, the at least 13 disulfide bonds formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: i and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3. In some embodiments, the 14 disulfide bonds are formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3. In some embodiments, an isolated recombinant antibody comprises a far UV circular dichroism peak at a wavelength between 200 nm and 205 nm. In some embodiments, an isolated recombinant antibody comprises a near UV circular dichroism peak at a wavelength between 270 nm and 290 nm. In some embodiments, the near UV circular dichroism peak is at a wavelength between 280 nm and 290 nm. In some embodiments, an isolated recombinant antibody comprises a T0 between 59 ℃ to 61 ℃, Tm1 is between 69 ℃ to 71 ℃, and Tm2 is between 71 ℃ to 74 ℃. In some embodiments, the T0 is 60.3 ℃, Tm1 is 70.0 ℃, and Tm2 is 74.1 ℃. In some embodiments, an isolated  recombinant antibody comprises at least one N-glycan moiety comprising N-acetylglucosamine (GlcNAc) , hexose, fucose, N-Acetylneuraminic acid (Neu5Ac) , or N-Glycolylneuraminic acid (Neu5Gc) . In some embodiments, the at least one N-gtycan moiety comprises GlcNAc, hexose, or Neu5Gc. In some embodiments, the at least one N-glycan moiety comprises GlcNAc and hexose. In some embodiments, the at least one N-glycan moiety comprises GlcNAc, hexose, and Neu5Gc. In some embodiments, the at least one N-glycan moiety comprises at least two GlcNAc moieties and at least two hexose moieties. In some embodiments, the at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises five GleNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAe moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glyean moiety comprises five GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, and one Neu5Ae moiety. In some embodiments, the at least one N-glycan moiety comprises two GlcNAc moieties and eight hexose moieties. In some embodiments, the at least one N-glycan moiety is at Asparagine 300 of SEQ ID NO: 2. In some embodiments, the at least one N-glycan moiety is at Asparagine 334 of SEQ ID NO: 3.
Disclosed herein are a plurality of isolated recombinant antibodies that target CD38 and ICAM1. In some embodiments, the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1. In some embodiments, the isolated recombinant antibodies comprise a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2. In some embodiments, the heavy chain hole (HC-Hole) amino acid sequence has at least 80%sequence identity to SEQ ID NO: 3. In some embodiments, a plurality comprises greater than 90%monomer of the isolated recombinant antibodies. In some embodiments, the plurality of isolated recombinant antibodies, comprise greater than 95%monomer. In some embodiments, the plurality of isolated recombinant antibodies, comprises greater than 99%monomer. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of greater than or equal to 1.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 2.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 5.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 10.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration of at least 15.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a concentration  of at least 20.0 mg/mL. In some embodiments, the plurality of isolated recombinant antibodies comprise a buffered solution having a pH less than or equal to 5.5. In some embodiments, the buffered solution comprises one or more of acetate, phosphate, or histidine. In some embodiments, the buffered solution comprises acetate at a concentration greater than 15mM. In some embodiments, the buffered solution comprises sucrose. In some embodiments, the plurality of isolated recombinant antibodies comprise sucrose at a concentration greater than or equal to 5%w/v. In some embodiments, the buffered solution comprises polysorbate 80. In some embodiments, polysorbate 80 is at a concentration greater than or equal to 0.01%w/v. In some embodiments, the plurality of isolated recombinant antibodies comprise greater than 90%monomer at a concentration of greater than or equal to 20.0 mg/mL in 20 mM acetate, 8%(w/v) sucrose, 0.02% (w/v) polysorbate 80, pH of 5.0.
Disclosed herein, are methods of treating a cancer in a subject in need thereof comprising administering to the subject the isolated recombinant antibody of any of the above embodiments or the plurality of isolated recombinant antibodies according to any of the above embodiments. In some embodiments, the cancer comprises a cell that expresses CD38 and ICAM1. In some embodiments, the cancer comprises a solid tumor or a hematological malignancy. In some embodiments, the cancer comprises the hematological malignancy. In some embodiments, the hematological malignancy is multiple myeloma, lymphoma, or Burkitt lymphoma. In some embodiments, the cancer is a lung cancer or a prostate cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor comprise a refractory solid tumor. In some embodiments, the solid tumor comprises a relapsed solid tumor. In some embodiments, the multiple myeloma comprises a refractory multiple myeloma. In some embodiments, the multiple myeloma comprises relapsed multiple myeioma. In some embodiments, the lymphoma comprises a refractory lymphoma. In some embodiments, the lymphoma comprises a relapsed lymphoma. In some embodiments, the subject has received a diagnosis of a relapsed or refractory solid tumor. In some embodiments, the subject has received a diagnosis of relapsed or refractory multiple myeloma. In some embodiments, the subject has received a diagnosis of relapsed or refractory lymphoma.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A, 1B, and 1C illustrate manufacturing and purification processes for an isolated recombinant antibody described herein.
FIG. 2 illustrates schematic representation of a 3-chain isolated recombinant antibody described herein that targets CD38 and ICAM1. Shown is a light chain (LC) , a heavy chain-knob (HC-Knob) , and a heavy chain-hole (HC-Hole) .
FIG. 3 illustrates theoretical disulfide bond linkages for an isolated recombinant antibody described herein.
FIG. 4 illustrates overlaid UV chromatograrns of non-reduced and reduced peptide maps of an isolated recombinant antibody described herein by Lys-C/trypsin sequential digestion with peak annotation.
FIG. 5 illustrates far-UV CD spectra of an isolated recombinant antibody described herein.
FIG. 6 illustrates near-UV CD spectra of an isolated recombinant antibody described herein.
FIG. 7 illustrates DSC thermogram of an isolated recombinant antibody described herein.
FIG. 8A illustrates a full-view chromatogram of N-glycans for an isolated recombinant antibody described herein
FIG. 8B illustrates a zoom-in chromatograrn of N-glycans of the recombinant antibody.
FIG. 9 illustrates the symbol structures of N-glycans.
DETAILED DESCRIPTION OF THE INVENTION
Antibodies that bind to CD38 are useful for the treatment of cancers that express CD38. Anti-CD38 antibodies are thought to kill cancer cells by various mechanisms including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. One such antibody, daratumumab, is approved for the treatment of adults with multiple myeloma. Reduced CD38 expression can limit the efficacy of anti-CD38 antibodies. Proposals to overcome this limitation include treatment with an antibody having a higher affinity for CD38, treatment with an antibody that binds to a different epitope on CD38, treatment with an antibody that more effectively inhibits CD38 enzymatic activity, treatment with a tetravalent anti-CD38 antibody, and concurrent treatment with all-trans retinoic acid to increase CD38 expression.
Antibody-based therapeutics have emerged as effective cancer treatment options due to the specificity and affinity of the antibody to target binding and ease of chemical and molecular modifications. For example, approved antibody-based therapy includes monoclonal antibodies such as rituximab, tositumomab, and trastuzumab; and bispecific T-cell engager such as Blinatumomab.
In some instances, low receptor copy numbers on a target cell, or low affinity toward an antigen has hindered the therapeutic effect of an antibody-based therapy. In some cases, non-specificity of an antibody toward a target antigen or the presence of a target in both cancer and non-cancer cells have further limited the use of an antibody-based therapy.
CD38, also known as cyclic ADP ribose hydrolase, is a type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 mediates cytokine secretion and activation and proliferation of lymphocytes (Funaro et al, J Immunology 145: 2390-6, 1990; Guse et al, Nature 398: 70-3, 1999) , and via its NAD glycohydrolase activity regulates extracellular NAD+ levels which have been implicated in modulating the regulatory T-cell compartment (Adriouch et al., 14: 1284-92, 2012; Chiarugi et al., Nature Reviews 12: 741-52, 2012) . In some instances, CD38 is upregulated in different types of cancer, in particular, in hematologic malignancies such as multiple myeloma.
ICAM1, also known as CD54, is an lg-like cell adhesion molecule. ICAM1 is an endothelial-and leukocyte-associated transmembrane protein and is involved in stabilizing cell-cell interactions and facilitating leukocyte endothelial transmigration. ICAM1 is expressed in various cell types, including endothelial cells and leukocytes, and can be expressed or overexpressed in different cancer cells such as  myeloma, pancreatic cancer, glioma, lung cancer, melanoma, colorectal cancer, and lymphoma.
Disclosed herein are isolated recombinant antibodies that target CD38 and ICAM1. In some embodiments, the isolated recombinant antibodies comprise beneficial characteristics related to secondary structure, tertiary structure, melting temperature, or post-translational modifications, that enhance efficacy or improve stability of the recombinant antibodies that target CD38 or ICAM1.
Disclosed herein are isolated recombinant antibodies that target CD38 and ICAM1 comprising a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3, and wherein the isolated recombinant antibody has at least one of the following characteristics: (a) at least one disulfide bond formed by a pair of cysteine residues; (b) a far UV circular dichroism peak at a wavelength between 195 nm and 210 nm when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; (c) a near UV circular dichroism peak at a wavelength between 270 nm and 300 nm when the isolated recombinant 3 antibody is formulated at a concentration of 1.0 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; (d) an onset melting temperature (To) between 58 ℃ to 62 ℃, a transition midpoint temperature 1 (Tm1) between 68 ℃ to 71 ℃, a transition midpoint temperature 2 (Tm2) between 72 ℃ to 75 ℃, when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; and (e) at least one N-glycan moiety.
In some embodiments, the isolated recombinant antibody comprises at least two of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least three of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least four of the characteristics. In some embodiments, the isolated recombinant antibody comprises at least five of the characteristics.
In some embodiments, the isolated recombinant antibody comprises two of the characteristics. In some embodiments, the isolated recombinant antibody comprises three of the characteristics. In some embodiments, the isolated recombinant antibody comprises four of the characteristics. In some embodiments, the isolated recombinant antibody comprises five of the characteristics.
Amino Acid Sequences of Isolated Recombinant Antibodies
In some embodiments, the LC comprises at least 85%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 90%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob  comprises at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 90%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO:3. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
In some embodiments, the HC-knob sequence does not comprise a C-terminal lysine 450. In some embodiments, the HC-knob sequence comprises an amidated C-terminal proline 448. In some embodiments the HC-knob sequence does not comprise a C-terminal lysine 450 and glycine 449.
In some embodiments, the HC-hole sequences does not comprise a C-terminal lysine 484. In some embodiments, the HC-hole sequence comprises an amidated C-terminal proline 482. In some embodiments the HC-hole sequence does not comprise a C-terminal lysine 484 and glycine 483.
In some embodiments, the HC-hole sequence comprises an oxidated methionine 289 or an oxidated methionine 465. In some embodiments, the HC-knob sequence comprises an oxidated methionine 255 or an oxidated methionine 431.
In some embodiments the HC-hole sequence comprises one or more ora deamidated asparagine 352, deamidated asparagine 421, and deamidated asparagine 465.
In some embodiments the HC-knob sequence comprises one or more of a deamidated asparagine 318, deamidated asparagine 387, and deamidated asparagine 437.
In some embodiments, the HC-knob sequences comprises a succinimide formation at asparagine 318 or asparagine 387. In some embodiments, the HC-knob sequences comprises a succinimide formation at asparagine 352 or asparagine 421.
Table 1. Exemplary Amino Acid Sequences of an Isolated Recombinant Antibody that Targets CD38 and ICAM1

Disulfide Bonds
In some embodiments, the at least one disulfide bond is an intrachain disulfide bond. In some embodiments, the at least one disulfide bond is an interchain disulfide bond. In some embodiments, the interchain disulfide bond is between the HC-Knob and the HC-Hole. In some embodiments, isolated recombinant antibody comprises at least two disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least three disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least four disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least five disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least six disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least seven disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least eight disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least nine disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least ten disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least eleven disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least twelve disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least thirteen disulfide bonds formed by pairs of cysteine residues. In some embodiments, isolated recombinant antibody comprises at least fourteen disulfide bonds formed by pairs of cysteine residues.
In some embodiments, the pair of cysteine residues comprises Cysteine 23 and Cysteine 88 of  SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 133 and Cysteine 197 of SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1. In some embodiments, the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 2. In some embodiments, the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3. In some embodiments, the pair of cysteine residues comprises Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
In some embodiments, the at least one disulfide bond formed by a pair of cysteine residues is selected from Cysteine 23 of SEQ ID NO: l and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
In some embodiments, the isolated recombinant antibody comprises at least seven disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
In some embodiments, the isolated recombinant antibody comprises at least ten disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
In some embodiments, the isolated recombinant antibody comprises at least 12 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
In some embodiments, the isolated recombinant antibody comprises at least 13 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of
SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
In some embodiments, the isolated recombinant antibody comprises 14 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1; Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1; Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1; Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2; Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2; Cysteine 229 of SEQ ID NO: 2 and  Cysteine 263 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3; Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3; Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2; Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2; Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3; Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
UV Dichroism Peaks and Melting Temperatures
In some embodiments, the far UV circular dichroism peak is at a wavelength between 200 nm and 205 nm. In some embodiments, the near UV circular dichroism peak is at a wavelength between 270 nm and 290 nm. In some embodiments, the near UV circular dichroism peak is at a wavelength between 280 nm and 290 nm.
In some embodiments, T0 is between 59 ℃ to 61 ℃, Tm1 is between 69 ℃ to 71 ℃, and Tm2 is between 71 ℃ to 74 ℃. The isolated recombinant antibody of any one of the above claims, wherein the T0 is 60.3 ℃, Tm1 is 70.0 ℃, and Tm2 is 74.1 ℃. In some embodiments, T0. Tm1, or Tm2 is measured by differential scanning calorimetry (DSC) .
N-glycan Moieties
In some embodiments, the recombinant three chain antibody comprises at least one N-glycan moiety. In some embodiments, the recombinant three chain antibody comprises at least two N-glycan moieties. In some embodiments the heavy chain knob sequence comprises at least one N-glycan moiety. In some embodiments the heavy chain known sequences comprises a N-glycan moiety at Asparagine 300. In some embodiments the heavy chain hole sequence comprises at least one N-glycan moiety. In some embodiments the heavy chain hole sequence comprises a N-glycan moiety at Asparagine 334.
In some embodiments, the at least one N-glycan moiety comprises N-acetylglucosamine (GlcNAc) , hexose, fucose, N-Acetylneuraminic acid (Neu5Ac) , or N-Glycolylneuraminic acid (Neu5Gc) . In some embodiments, the at least one N-glycan moiety comprises GlcNAc, hexose, or Neu5Gc. In some embodiments, the at least one N-glycan moiety comprises GlcNAc and hexose. In some embodiments, the at least one N-glycan moiety comprises GlcNAc, hexose, and Neu5Gc. In some embodiments, the at least one N-glycan moiety comprises at least two GlcNAc moieties and at least two hexose moieties. In some embodiments, the at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises five GlcNAc moieties and three hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises  five GlcNAc moieties and four hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties. In some embodiments, the at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, and one Neu5Ac moiety. In some embodiments, the at least one N-glycan moiety comprises two GlcNAc moieties and eight hexose moieties.
Formulations of Isolated Recombinant Antibodies that Target CD38 and ICAM1
Disclosed herein includes formulation (s) comprising a population of antibodies or recombinant antibodies, such as comprising any one or a combination the antibodies or recombinant antibodies as described herein.
Disclosed herein is a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, wherein the one or more pharmaceutically acceptable excipients comprise histidine, sucrose, polysorbate-20, sodium phosphate, citrate, acetate, sodium chloride, potassium chloride, magnesium chloride, and calcium chloride. Disclosed herein is a pharmaceutical composition, wherein the pharmaceutical composition comprises the recombinant polypeptide (such as any described herein) , sodium phosphate monobasic monohydrate, sodium phosphate dibasic, citrate, acetate, histidine, heptahydrate, sodium chloride, potassium chloride, histidine, citrate, acetate, sucrose, polysorbate-20, polysorbate 80, magnesium chloride hexahydrate and calcium chloride dihydrate. The pharmaceutical composition can have a pH less than or equal to 6.5, 6.0, 5.5, 5.0, or 4.5. The pharmaceutical composition can have a pH greater than or equal to 4.5, 5.0, 5.5, 6.0 or 6.5. The pharmaceutical composition can have a pH between 4.0 and 5.0, between 4.5 and 5.5, or between 5.0 and 6.0. The pharmaceutical composition can comprise greater than or equal to 10mM, 11 mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, or 25mM Acetate. The pharmaceutical composition can comprise less than or equal to 25mM, 24mM, 23mM, 22mM, 21 mM, 20mM, 19mM, 18mM, 17mM, 16mM, 15mM, 14mM, 13mM, 12mM, 1 lmM, or 10mM Acetate. The pharmaceutical composition can comprise greater than or equal to 10mM, 1 lmM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, or 25mM Histidine. The pharmaceutical composition can comprise less than or equal to 25mM, 24mM, 23mM, 22mM, 21mM, 20mM, 19mM, 18mM, 17mM, 16mM, 15mM, 14mM, 13mM, 12mM, 1 lmM, or 10mM Histidine. The pharmaceutical composition can comprise greater than or equal to 5% (w/v) , 6% (w/v) , 7% (w/v) , 8% (w/v) , 9% (w/v) , or 10% (w/v) sucrose. The pharmaceutical composition can comprise less than or equal to 10% (w/v) , 9% (w/v) , 8% (w/v) , 7% (w/v) , 6% (w/v) , or 5% (w/v) sucrose. The pharmaceutical composition can comprise less than or equal to 0.05% (w/v) polysorbate 80, 0.04% (w/v) polysorbate 80, 0.03% (w/v) polysorbate 80, 0.02% (w/v) polysorbate 80, or 0.01%polysorbate 80. The pharmaceutical composition can comprise greater than or equal to 0.01% (w/v) polysorbate 80, 0.02% (w/v) polysorbate 80, 0.03% (w/v) polysorbate 80, 0.04% (w/v) polysorbate 80, or 0.05%polysorbate 80. Disclosed herein is a pharmaceutical composition, wherein the pharmaceutical composition comprises the recombinant polypeptide (such as any described herein) , 20 mM Acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0.
In some embodiments of the formulation, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,  98%, or 99% (e.g., by mole or by mass) of the antibodies of the population is monomeric. In some embodiments of the formulation, less than or equal to 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% (e.g., by mole or by mass) of the antibodies of the population is aggregated.
Disclosed herein are a plurality of isolated recombinant antibodies that target CD38 and ICAM1 wherein the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3 wherein the plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
In some embodiments, the plurality comprises greater than 91%monomer. In some embodiments, the plurality comprises greater than 92%monomer. In some embodiments, the plurality comprises greater than 93%monomer. In some embodiments, the plurality comprises greater than 94%monomer. In some embodiments, the plurality comprises greater than 95%monomer. In some embodiments, the plurality comprises greater than 96%monomer. In some embodiments, the plurality comprises greater than 97%monomer. In some embodiments, the plurality comprises greater than 98%monomer. In some embodiments, the plurality comprises greater than 99%monomer.
In some embodiments, the LC comprises at least 85%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 90%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 95%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1. In some embodiments, the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2. In some embodiments, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2. In some embodiments, the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 90%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3. In some embodiments, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3. In some embodiments, the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
In some embodiments, the concentration of the isolated recombinant antibodies is greater than or equal to 1.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at  least 2.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 5.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 10.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 15.0 mg/mL. In some embodiments, the concentration of the isolated recombinant antibodies is at least 20.0 mg/mL. In some embodiments, the plurality is in a buffered solution having a pH less than or equal to 5.5. In some embodiments, the buffered solution comprises one or more of acetate, phosphate, or histidine. In some embodiments, the buffered solution comprises acetate at a concentration greater than 15mM. In some embodiments, the buffered solution comprises sucrose. In some embodiments, the sucrose is at a concentration greater than or equal to 5%w/v. In some embodiments, the buffered solution comprises polysorbate 80. In some embodiments, the polysorbate 80 is at a concentration greater than or equal to 0.01%w/v. In some embodiments, the plurality comprises greater than 90%monomer at a concentration of greater than or equal to 20.0mg/mL in 20 mM acetate, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH of 5.0.
Kits
Provided herein, in some embodiments, is a kit comprising a recombinant antibody (such as any described herein) or a composition (such as any described herein) , a container, and a label or package insert on or associated with the container.
Methods of Treatment
In some embodiments are methods of treating a subject having cancer, the method comprising: administering to the subject any of the antibodies that bind specifically to CD38 and ICAM1 as disclosed herein. In some embodiments, the cancer comprises cancer cells that express CD38 or ICAM1. In some embodiments, the cancer cells that express CD38 or ICAM1 are lysed. In some embodiments, the antibody induces antibody-dependent cellular phagocytosis (ADCP) of the cancer cells that express CD38 or ICAM1. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is B cell cancer. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the hematological malignancy comprises a multiple myeloma. In some embodiments, the multiple myeloma comprises refractory multiple myeloma. In some embodiments, the multiple myeloma comprises relapsed multiple myeloma. In some embodiments, the cancer is lymphoma, and wherein the lymphoma is B-cell lymphoma. In some embodiments, the cancer is lymphoma, and wherein the lymphoma is T-cell lymphoma. In some embodiments, the lymphoma comprises refractory lymphoma. In some embodiments, the lymphoma comprises relapsed lymphoma. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor comprises a refractory solid tumor. In some embodiments, the solid tumor comprises a relapsed solid tumor. In some embodiments, the solid tumor is sarcoma, breast cancer, lung cancer, carcinoma, ovarian cancer, pancreatic cancer, gastric cancer, colorectal cancer, endometrial cancer, esophageal cancer, prostate cancer, cervical cancer, kidney cancer, urothelial cancer, or head and neck cancer. In some embodiments, the solid tumor is lung cancer, and wherein the lung cancer is non-small cell lung cancer. In some embodiments, the solid tumor is lung cancer, and wherein the lung cancer is small cell lung cancer. In  some embodiments, the solid tumor is a sarcoma. In some embodiments, the solid tumor is breast cancer. In some embodiments, the solid tumor is a carcinoma. In some embodiments, the solid tumor is ovarian cancer. In some embodiments, the solid tumor is pancreatic cancer. In some embodiments, the solid tumor is gastric cancer. In some embodiments, the solid tumor is colorectal cancer. In some embodiments, the solid tumor is head and neck cancer.
In some embodiments, the method further comprises administering to the subject an anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent or a biologic agent. In some embodiments, the administering is sufficient to reduce or eliminate the cancer as compared to a comparable method lacking the administering.
Articles of Manufacture
In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper that is pierceable by a hypodermic injection needle) . At least one active agent in the composition is an antibody that specifically binds to CD38 and ICAM1.
The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises the bispecific antibody of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI) , phosphate-buffered saline, Ringer′ssolution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Certain Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a, ” “an, ” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include, ” “includes, ” and “included, ” is not limiting.
As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 μL” means “about 5 μL” and also “5 μL. ” Generally, the term “about” includes an amount that would be expected to be within experimental error.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
“Antibodies” and “immunoglobulins” (IGs) are glycoproteins having the same structural characteristics. The terms are used synonymously. In some instances, the antigen specificity of the immunoglobulin is known.
The term “antibody” is used in the broadest sense, and covers fully assembled antibodies, antibody fragments that can bind antigen (e.g., Fab, F (ab’ ) 2, Fy, single chain antibodies (scFv) , diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, and the like) , and recombinant peptides comprising the forgoing.
As used herein, the terms “individual (s) , ” “subject (s) , ” and “patient (s) ” mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker) .
As used herein, the term “percent (%) amino acid sequence identity” with respect to a sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as EMBOSS MATCHER, EMBOSS WATER, EMBOSS STRETCHER, EMBOSS NEEDLE, EMBOSS LALIGN, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the %amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain %amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program′s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the %amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically stated otherwise, all %amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
The terms “individual (s) ” , “subject (s) ” and “patient (s) ” are used interchangeably herein and refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker) .
EMBODIMENTS
Embodiment 1. An isolated recombinant antibody that targets CD38 and ICAM1 comprising a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3, and wherein the isolated recombinant antibody has at least one of the following characteristics:
(a) at least one disulfide bond formed by a pair of cysteine residues;
(b) a far UV circular dichroism peak at a wavelength between 195 nm and 210 nm when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0;
(c) a near UV circular dichroism peak at a wavelength between 270 nm and 300nm when the isolated recombinant 3 antibody is formulated at a concentration of 1.0 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0;
(d) an onset melting temperature (T0) between 58 ℃ to 62 ℃, a transition midpoint temperature 1 (Tm1) between 68 ℃ to 71 ℃, a transition midpoint temperature 2 (Tm2) between 72 ℃ to 75 ℃, when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; or
(e) at least one N-glycan moiety.
Embodiment 2. The isolated recombinant antibody of embodiment 1, wherein the LC comprises at least 85%sequence identity to SEQ ID NO: 1.
Embodiment 3. The isolated recombinant antibody of any one of embodiments 1-2, wherein the LC comprises at least 90%sequence identity to SEQ ID NO: 1.
Embodiment 4. The isolated recombinant antibody of any one of embodiments 1-3, wherein the LC comprises at least 95%sequence identity to SEQ ID NO: 1.
Embodiment 5. The isolated recombinant antibody of any one of embodiments 1-4, wherein the LC comprises at least 99%sequence identity to SEQ ID NO: 1.
Embodiment 6. The isolated recombinant antibody of any one of embodiments 1-5, wherein the LC comprises the amino acid sequence according to SEQ ID NO: 1.
Embodiment 7. The isolated recombinant antibody of any one of embodiments 1-6, wherein the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2.
Embodiment 8. The isolated recombinant antibody of any one of embodiments 1-7, wherein the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2.
Embodiment 9. The isolated recombinant antibody of any one of embodiments 1-8, wherein the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2.
Embodiment 10. The isolated recombinant antibody of any one of embodiments 1-9, wherein the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2.
Embodiment 11. The isolated recombinant antibody of any one of embodiments 1-10, wherein the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2.
Embodiment 12. The isolated recombinant antibody of any one of embodiments 1-11 wherein the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3.
Embodiment 13. The isolated recombinant antibody of any one of embodiments 1-12, wherein the HC-Hole comprises at least 90%sequence identity to SEQ ID NO: 3.
Embodiment 14. The isolated recombinant antibody of any one of embodiments 1-13, wherein the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3.
Embodiment 15. The isolated recombinant antibody of any one of embodiments 1-14, wherein the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3.
Embodiment 16. The isolated recombinant antibody of any one of embodiments 1-15, wherein the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
Embodiment 17. The isolated recombinant antibody of any one of embodiments 1-16, wherein the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2, HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
Embodiment 18. The isolated recombinant antibody of any one of embodiments 1-17, wherein the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
Embodiment 19. The isolated recombinant antibody of any one of embodiments 1-18, wherein the isolated recombinant antibody comprises at least two of the characteristics.
Embodiment 20. The isolated recombinant antibody of any one of embodiments 1-19, wherein the isolated recombinant antibody comprises at least three of the characteristics.
Embodiment 21. The isolated recombinant antibody of any one of embodiments 1-20, wherein the isolated recombinant antibody comprises at least four of the characteristics.
Embodiment 22. The isolated recombinant antibody of any one of embodiments 1-21, wherein the isolated recombinant antibody comprises at least five of the characteristics.
Embodiment 23. The isolated recombinant antibody of any one of embodiments 1-22, wherein the at least one disulfide bond is an intrachain disulfide bond.
Embodiment 24. The isolated recombinant antibody of any one of embodiments 1-23, wherein the at least one disulfide bond is an interchain disulfide bond.
Embodiment 25. The isolated recombinant antibody of any one of embodiments 1-24, wherein the interchain disulfide bond is between the HC-Knob and the HC-Hole.
Embodiment 26. The isolated recombinant antibody of any one of embodiments 1-25, wherein the isolated recombinant antibody comprises at least two disulfide bonds formed by pairs of cysteine residues.
Embodiment 27. The isolated recombinant antibody of any one of embodiments 1-26, wherein the isolated recombinant antibody comprises at least three disulfide bonds formed by pairs of cysteine residues.
Embodiment 28. The isolated recombinant antibody of any one of embodiments 1-27, wherein the isolated recombinant antibody comprises at least four disulfide bonds formed by pairs of cysteine residues.
Embodiment 29. The isolated recombinant antibody of any one of embodiments 1-28, wherein the isolated recombinant antibody comprises at least five disulfide bonds formed by pairs of cysteine residues.
Embodiment 30. The isolated recombinant antibody of any one of embodiments 1-29, wherein the isolated recombinant antibody comprises at least six disulfide bonds formed by pairs of cysteine residues.
Embodiment 31. The isolated recombinant antibody of any one of embodiments 1-30, wherein the isolated recombinant antibody comprises at least seven disulfide bonds formed by pairs of cysteine residues.
Embodiment 32. The isolated recombinant antibody of any one of embodiments 1-31, wherein the isolated recombinant antibody comprises at least eight disulfide bonds formed by pairs of cysteine residues.
Embodiment 33. The isolated recombinant antibody of any one of embodiments 1-32, wherein the isolated recombinant antibody comprises at least nine disulfide bonds formed by pairs of cysteine residues.
Embodiment 34. The isolated recombinant antibody of any one of embodiments 1-33, wherein the isolated recombinant antibody comprises at least ten disulfide bonds formed by pairs of cysteine residues.
Embodiment 35. The isolated recombinant antibody of any one of embodiments 1-34, wherein the isolated recombinant antibody comprises at least eleven disulfide bonds formed by pairs of cysteine  residues.
Embodiment 36. The isolated recombinant antibody of any one of embodiments 1-35, wherein the isolated recombinant antibody comprises at least twelve disulfide bonds formed by pairs of cysteine residues.
Embodiment 37. The isolated recombinant antibody of any one of embodiments 1-36, wherein the isolated recombinant antibody comprises at least thirteen disulfide bonds formed by pairs of cysteine residues.
Embodiment 38. The isolated recombinant antibody of any one of embodiments 1-37, wherein the isolated recombinant antibody comprises at least fourteen disulfide bonds formed by pairs of cysteine residues.
Embodiment 39. The isolated recombinant antibody of any one of embodiments 1-38, wherein the pair of cysteine residues comprises Cysteine 23 and Cysteine 88 of SEQ ID NO: 1.
Embodiment 40. The isolated recombinant antibody of any one of embodiments 1-39, wherein the pair of cysteine residues comprises Cysteine 133 and Cysteine 197 of SEQ ID NO: 1.
Embodiment 41. The isolated recombinant antibody of any one of embodiments 1-40, wherein the pair of cysteine residues comprises Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1.
 Embodiment 42. The isolated recombinant antibody of any one of embodiments 1-41, where1n the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2.
Embodiment 43. The isolated recombinant antibody of any one of embodiments 1-42, wherein the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 2.
Embodiment 44. The isolated recombinant antibody of any one of embodiments 1-43, wherein the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3.
Embodiment 45. The isolated recombinant antibody of any one of embodiments 1-44, wherein the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3.
Embodiment 46. The isolated recombinant antibody of any one of embodiments 1-45, wherein the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3.
Embodiment 47. The isolated recombinant antibody of any one of embodiments 1-46, wherein the pair of cysteine residues comprises Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 3.
Embodiment 48. The isolated recombinant antibody of any one of embodiments 1-47, wherein the pair of cysteine residues comprises Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 3.
Embodiment 49. The isolated recombinant antibody of any one of embodiments 1-48, wherein the pair of cysteine residues comprises Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3.
Embodiment 50. The isolated recombinant antibody of any one of embodiments 1-49, wherein the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3.
Embodiment 51. The isolated recombinant antibody of any one of embodiments 1-50, wherein the pair of cysteine residues comprises Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
Embodiment 52. The isolated recombinant antibody of any one of embodiments 1-51, wherein the at least one disulfide bond formed by a pair of cysteine residues is selected from
Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
Embodiment 53. The isolated recombinant antibody of any one of embodiments 1-52, wherein the isolated recombinant antibody comprises at least seven disulfide bonds formed by a pair of cysteine residues selected from
Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
Cysteine 137 of SEQ ID NO: I and Cysteine 197 of SEQ ID NO: 1;
Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
Embodiment 54. The isolated recombinant antibody of any one of embodiments 1-53, wherein the isolated recombinant antibody comprises at least ten disulfide bonds formed by a pair of cysteine residues selected from
Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
Cysteine 223 of S EQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
Embodiment 55. The isolated recombinant antibody of any one of embodiments 1-54, wherein the isolated recombinant antibody comprises at least 12 disulfide bonds formed by a pair of cysteine residues selected from
Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
Embodiment 56. The isolated recombinant antibody of any one of embodiments 1-55, wherein  the isolated recombinant antibody comprises at least 13 disulfide bonds formed by a pair of cysteine residues selected from
Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
Embodiment 57. The isolated recombinant antibody of any one of embodiments 1-56, wherein the isolated recombinant antibody comprises 14 disulfide bonds formed by a pair of cysteine residues selected from Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
Embodiment 58. The isolated recombinant antibody of any one of embodiments 1-57, wherein the far UV circular dichroism peak is at a wavelength between 200 nm and 205 nm.
Embodiment 59. The isolated recombinant antibody of any one of embodiments 1-58, wherein the near UV circular dichroism peak is at a wavelength between 270 nm and 290 nm.
Embodiment 60. The isolated recombinant antibody of any one of embodiments 1-59, wherein the near UV circular dichroism peak is at a wavelength between 280 nm and 290 nm.
Embodiment 61. The isolated recombinant antibody of any one of embodiments 1-60, wherein T0 is between 59 ℃ to 61 ℃, Tm1 is between 69 ℃ to 71 ℃, and Tm2 is between 71 ℃ to 74 ℃.
Embodiment 62. The isolated recombinant antibody of any one of embodiments 1-61, wherein the T0is 60.3 ℃, Tm1 is 70.0 ℃, and Tm2 is 74.1 ℃.
Embodiment 63. The isolated recombinant antibody of any one of embodiments 1-62, wherein the at least one N-glycan moiety comprises N-acetylglucosamine (GlcNAc) , hexose, fucose, N-
Acetylneuraminic acid (Neu5Ac) , or N-Glycolylneuraminic acid (Neu5Gc) .
Embodiment 64. The isolated recombinant antibody of any one of embodiments 1-63, wherein the at least one N-glycan moiety comprises GlcNAc, hexose, or Neu5Gc.
Embodiment 65. The isolated recombinant antibody of any one of embodiments 1-64, wherein the at least one N-glycan moiety comprises GlcNAc and hexose.
Embodiment 66. The isolated recombinant antibody of any one of embodiments 1-65, wherein the at least one N-glycan moiety comprises GlcNAc, hexose, and Neu5Gc.
Embodiment 67. The isolated recombinant antibody of any one of embodiments 1-66, wherein the at least one N-glycan moiety comprises at least two GlcNAe moieties and at least two hexose moieties.
Embodiment 68. The isolated recombinant antibody of any one of embodiments 1-67, wherein the at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties.
Embodiment 69. The isolated recombinant antibody of any one of embodiments 1-68, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties.
Embodiment 70. The isolated recombinant antibody of any one of embodiments 1-69, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties.
Embodiment 71. The isolated recombinant antibody of any one of embodiments 1-70, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties.
Embodiment 72. The isolated recombinant antibody of any one of embodiments 1-71, wherein the at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties.
Embodiment 73. The isolated recombinant antibody of any one of embodiments 1-72, wherein the at least one N-glycan moiety comprises five GlcNAc moieties and three hexose moieties.
Embodiment 74. The isolated recombinant antibody of any one of embodiments 1-73, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and four hexose moieties.
Embodiment 75. The isolated recombinant antibody of any one of embodiments 1-74, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties.
Embodiment 76. The isolated recombinant antibody of any one of embodiments 1-75, wherein the at least one N-glycan moiety comprises five GlcNAc moieties and four hexose moieties.
Embodiment 77. The isolated recombinant antibody of any one of embodiments 1-76, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties.
Embodiment 78. The isolated recombinant antibody of any one of embodiments 1-77, wherein  the at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, and one Neu5Ac moiety.
Embodiment 79. The isolated recombinant antibody of any one of embodiments 1-78, wherein the at least one N-glycan moiety comprises two GlcNAc moieties and eight hexose moieties.
Embodiment 80. The isolated recombinant antibody of any one of embodiments 1-79, wherein the at least one N-glycan moiety is at Asparagine 300 of SEQ ID NO: 2.
Embodiment 81. The isolated recombinant antibody of any one of embodiments 1-80, wherein the at least one N-glycan moiety is at Asparagine 334 of SEQ ID NO: 3.
Embodiment 82. A plurality of isolated recombinant antibodies that target CD38 and ICAM1 wherein the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3 wherein the plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
Embodiment 83. The plurality of isolated recombinant antibodies of embodiment 82, comprising greater than 95%monomer.
Embodiment 84. The plurality of isolated recombinant antibodies of embodiment 82, comprising greater than 99%monomer.
Embodiment 85. The plurality of isolated recombinant antibodies of any one of embodiments 82-84, wherein the concentration of the isolated recombinant antibodies is greater than or equal to 1.0 mg/mL.
Embodiment 86. The plurality of isolated recombinant antibodies of any one of embodiments 82-86, wherein the concentration of the isolated recombinant antibodies is at least 2.0 mg/mL.
Embodiment 87. The plurality of isolated recombinant antibodies of any one of embodiments 82-86, wherein the concentration of the isolated recombinant antibodies is at least 5.0 mg/mL.
Embodiment 88. The plurality of isolated recombinant antibodies of any one of embodiments 82-87, wherein the concentration of the isolated recombinant antibodies is at least 10.0 mg/mL.
Embodiment 89. The plurality of isolated recombinant antibodies of any one of embodiments 82-88 wherein the concentration of the isolated recombinant antibodies is at least 15.0 mg/mL.
Embodiment 90. The plurality of isolated recombinant antibodies of any one of embodiments 82-89, wherein the concentration of the isolated recombinant antibodies is at least 20.0 mg/mL.
Embodiment 91. The plurality of isolated recombinant antibodies of any one of embodiments 82-90, wherein the plurality is in a buffered solution having a pH less than or equal to 5.5.
Embodiment 92. The plurality of isolated recombinant antibodies of any one of embodiments 82-91, wherein the buffered solution comprises one or more of acetate, phosphate, or histidine.
Embodiment 93. The plurality of isolated recombinant antibodies of any one of embodiments 82-92 wherein the buffered solution comprises acetate at a concentration greater than 15mM.
Embodiment 94. The plurality of isolated recombinant antibodies of any one of embodiments  82-93, wherein the buffered solution comprises sucrose.
Embodiment 95. The plurality of isolated recombinant antibodies of embodiment 94, wherein the sucrose is at a concentration greater than or equal to 5%w/v.
Embodiment 96. The plurality of isolated recombinant antibodies of any one of embodiments 82-95, wherein the buffered solution comprises polysorbate 80.
Embodiment 97. The plurality of isolated recombinant antibodies of embodiment 96, wherein the polysorbate 80 is at a concentration greater than or equal to 0.01%w/v.
Embodiment 98. The plurality of isolated recombinant antibodies of any one of embodiments 82-97, comprising greater than 90%monomer at a concentration of greater than or equal to 20.0mg/mL in 20 mM acetate, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH of 5.0.
Embodiment 99. A method of treating a cancer in a subject in need thereof comprising administering to the subject the isolated recombinant antibody of any one of embodiments 1-80, or the plurality of isolated recombinant antibodies of embodiments 81-97, wherein the cancer comprises a cell that expresses CD38 and ICAM1.
Embodiment 100. The method of embodiment 99, wherein the cancer comprises a solid tumor or a hematological malignancy.
Embodiment 101. The method of embodiment 99, wherein the cancer comprises the hematological malignancy.
Embodiment 102. The method of embodiment 101, wherein the hematological malignancy is multiple myeloma, lymphoma, or Burkitt lymphoma.
Embodiment 103. The method of embodiment 99, wherein the cancer is a lung cancer or a prostate cancer.
Embodiment 104. The method of embodiment 100, wherein the solid tumor comprises a refractory solid tumor.
Embodiment 105. The method of embodiment 100, wherein the solid tumor comprises a relapsed solid tumor.
Embodiment 106. The method of embodiment 102, wherein the multiple myeloma comprises refractory multiple myeloma.
Embodiment 107. The method of embodiment 102, wherein the multiple myeloma comprises relapsed multiple myeloma.
Embodiment 108. The method of embodiment 102, wherein the lymphoma comprises refractory lymphoma.
Embodiment 109. The method of embodiment 102, wherein the lymphoma comprises relapsed lymphoma.
Embodiment 110. The method of embodiment 102, wherein the subject has received a diagnosis of relapsed or refractory solid tumor.
Embodiment 110. The method of embodiment 102, wherein the subject has received a diagnosis of relapsed or refractory multiple myeloma.
Embodiment 110. The method of embodiment 102, wherein the subject has received a diagnosis of relapsed or refractory lymphoma.
EXAMPLES
The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art
Example 1: Manufacturing and purification of the drug substance
Cell culture: inoeulum expansion
Thaw of cell bank vial and expansion in shake flask (250 ml)
The drug manufacturing and purification process is a multi-step cell culture inoculation and expansion rounds. At each round of the cell expansion process, and prior to the next inoculum cycle, CHO cells were monitored for viability and cell density. An exemplary flow diagram showing the manufacturing and purification process that can be used to develop the drug substance disclosed herein is depicted in exemplary FIG. 1.
To initiate the inoculum expansion, an MCB vial from the cell bank was thawed, and cells were seeded in 50 ml Basal Medium 1 (Growth medium) as a starter culture and cultivated in 250 mL shake flasks at 37℃ (36.5℃ -37.5℃) growth parameters for two days. Following the two-day expansion, cells were monitored for viability and cell density (VCD) .
Inoculura expansion in shaker flasks (1 L)
The cells from the starter culture in the previous step were expanded by sub-cultivation in Basal Medium 1 (growth medium) in 1 mL shake flasks for approximately 3 days until sufficient cells were generated to inoculate the next sub-cultivation. Cells were propagated at the same growth temperature parameters as before. Cells were monitored for VCD.
Inoculum expansion in shake fiasks (2.8 L) :
For the next inoculum expansion, cells from the previous step were sub-cultivated in Basal medium 1 (growth medium) in 2.8 L shake flasks and propagated at the same growth temperature parameters for approximately 3 days until sufficient cells were generated to inoculate the 20 L Wave bioreactor. Cells were assessed for VCD.
Inoculum expansion in 1st stage inoculum bioreactor (20 L)
At the 1st stage of expansion in a bioreactor, an inoculum of cells from the 2.8 L culture expansion were transferred into a 20 L Wave bioreactor containing Basal medium 2 (growth medium) and cultivated at the same growth temperature parameters for approximately 3 days. Cells were assessed for VCD.
Inoculum expansion in 2nd stage inoculum bioreactor (100 L)
The 2nd stage bioreactor cell expansion was initiated by transferring cells from the previous 1st stage bioreactor cell expansion into a 100 L bioreactor containing Growth medium. Cells were cultivated at the same growth temperature parameters for approximately 3 days. Cells were assessed for VCD.
Cell Culture in Production Bioreactor (500 L)
Cell production in 500 rnL Bioreactor
At the production stage, the cell culture from the 2nd stage inoculum bioreactor was transferred to a 500 L production bioreactor containing growth medium supplemented with antifoam poloxamer. Cells were propagated at the same growth temperature parameter of 37 ℃ (36.5 -37.5 ℃) for either (a) 72 ± 4 hours post inoculation or (b) until the VCD reached (5.00 -10.00) x 106 cells/mL, whichever occurred first, and which signaled to shift or drop growth temperature parameters down to 33.0 ℃ (32.5 -33.5 ℃) .
The 500 L production bioreactor was operated in fed-batch mode with a total incubation time of about 14 days. For each production process, two different feeds and glucose were added aseptically to the bioreactor. The first feed (Feed 1) was Cell Boost 7a Feed Medium while the second feed (Feed 2) was Cell Boost 7b Feed Medium. The contents of the production bioreactor were harvested on day 14 or within 24 hours of a drop in viability below 60.0%, whichever occurred first. The unprocessed bulk (UPB) was evaluated for the bioburden, which included testing for mycoplasma, adventitious viruses in vitro, retroviruses, and species-specific viruses.
Purification and Modification Reactions
Harvest
The first step in the harvest and clarification process was to isolate cells and cell debri from the drug product by filtration. The harvest was carried out by depth filtration using filters STAX PDP8 and STAX PDD1 and capacities of≤ 56 L/m2 (primary) and ≤ 112 L/m2 (secondary) which was followed by 0.2 um filtration. The filtrate was collected in an agitated tank and maintained at room temperature. The filtrate was evaluated for bioburden and endotoxin contaminants.
Protein A Affinity Chromatography
The Protein A affinity step was operated in bind and elute mode and was used to capture the product while allowing impurities such as host cell proteins (HCP) , DNA, and medium additives to flow through. To start the purification, the processed harvest was loaded directly onto the Protein A column at a load temperature of 15 -26 ℃ and a loading capacity of 20 -40 g/L. The column was then washed, and the bound product was eluted with a low pH buffer. Product pooling was done by optical density (OD) A280 using Amsphere A3 as the resin. The filtrate was evaluated for bioburden and endotoxin contaminants.
Low pH Viral Inactivation and Neutralization
The low pH eluate from the Protein A step above was adjusted to pH 3.60 (3.50 -3.70) with 1M Acetic Acid and held for 60, ＜ 120 minutes at 15 -26℃ for viral inactivation and neutralization. At post-neutralization, the eluate was adjusted to pH 5.50 (5.40 -5.60) and held for 60 minutes with 1M Tris  base. The eluate was then assessed for bioburden and endotoxin contaminants.
Intermediate Depth Filtration
The pH adjusted Protein A eluate from the previous step, was filtered through the PDD1 depth filter which had been equilibrated using 30 mM Sodium Acetate at a load pH 5.5 and temperature of 15 -26 ℃ and washed. Filtration through the PDD1 depth filter used a nominal pore size of＜ 0.5 μm, a load capacity of ≤ 1196 g/m2 and load conductivity of 2.1mS/cm (1.9 -2.5 mS/em) . The eluate was evaluated for bioburden and endotoxin contaminant. Next, the intermediate depth filter pool was then filtered through a 0.2 μm filter.
Anion Exchange (AEX) Chromatography
The AEX chromatography operation was performed using the flow-through mode where potential virus contaminants and impurities were bound to the column while the product flowed through. The AEX column was equilibrated, and the intermediate depth filter pool was loaded at a temperature of 15 -26 ℃, loading capacity of 80 -169 g/L, and then collected as a flow through elution pool. The elution pool was washed with equilibration buffer and pooled by OD A280 using the Praesto Q65 resin. The filtrate was evaluated for bioburden and endotoxin contaminants. Next, the AEX pool was next filtered through a 0.2 μm sterile filter.
Cation Exchange (CEX) Chromatography
The filtered AEX pool was further purified over a cation exchange chromatography (CEX) . The CEX chromatography operation was performed in bind and elute mode. The column was equilibrated with a NaCl buffer at a load pH of 5.50 (5.30 -5.70) and load temperature of 15 -26 OC, and then loaded with the AEX pool. The CEX column was then washed, and the product was step eluted with a NaC1 buffer at the same load condition, with pooling by OD A280 using Capto SP ImpRes as a resin. The filtrate was evaluated for bioburden and endotoxin contaminants. In the next step, the resultant eluate was filtered through a 0.2 μm filter to remove viral contaminant.
Virus Filtration
The CEX pool eluted from the previous CEX procedure was adjusted for conductivity as necessary, with WFI to conductivity ≤ 7 mS/cm and then filtered through a Viresolve Magnus 2.2 shield pre-filter, followed by a Planova 20N virus nanofilter with a filter capacity at ≤ 968.6 g/m2, ≤ 102.7 L/m2 and operating pressure of 0.8 bar (0.7 -1.0 bar) .
The virus filter pool was then filtered through a 0.2μm filter. The Planova 20N nanofilter was tested for integrity prior to and after virus filtration and was additionally tested for bioburden and endotoxin contaminants.
Ultrafiltration and Diafiltration (UF/DF)
The eluate from the virus filtration pool was next diluted with formulation buffer to a target protein concentration of 20.0 g/L. Polysorbate 80 was added to a final concentration of 0.02% (w/v) . The concentrated eluate from the virus filtration pool was then processed through a buffer exchange into diafiltration buffer using Pellicon 3 Biomax 30kD molecular weight cut off UF/DF membranes, at a membrane capacity of ≤ 500 g/m2. The UF/DF pool was evaluated for bioburden, endotoxin  contaminants and for protein concentration. In the next procedure, the UF/DF pool was filtered through a 0.2μm filter.
Formulation
The UF/DF pool was diluted to the target concentration of 20.0 g/L with diafiltration buffer. Polysorbate 80 was added to a final concentration of 0.02% (w/v) . The pool was further tested for bioburden, endotoxin contaminants and protein concentration. The formed the drug substance was then filtered and packaged as described below. The formulation buffer was 20 mM Acetate, 8%sucrose, 0.02%PS80, pH 5.0.
Filling and Storage
The formulated drug substance from the previous steps was filtered through a 0.2 μm filter that had been tested for integrity. The drug substance was dispensed or filled into 1 L PETG (polyethylene terephthalate glycol) bottles in an appropriate number of bottles according to specified or required volumes. Following sterile filtration, integrity testing was performed on the filters and the drug substance was tested for bioburden, endotoxin contaminants and for protein concentration.
Example 2: Disulfide Bond Linkage Confirmation by LC-MS/MS
Drug molecule
The drug molecule is a bispecific antibody (BsAb) . The molecule consists of one light chains (LC) and two distinct heavy chains (HC-Knob and HC-Hole) . The schematic representation of the molecule is shown in FIG. 2. LC is kappa sub-type light chain and HC-Knob is immunoglobulin G1 (IgG1) sub-type heavy chain. HC-Hole consists of a scFv (single-chain variable fragment) and CH2 (constant heavy chain 2) domain, CH3 (constant heavy chain 3) domain of IgG1 sub-type heavy chain. For the scFv, a scFv-VL (light chain variable region) and a scFv-VH (heavy chain variable region) are connected with a (G4S) 3 linker. In addition, the “knob-into-hole” approach is adopted to tackle the problem of the homodimerization by substituting a large amino acid for a small one in the CH3 domain (the “knob” ) of HC-Knob and vice versa (the “hole” ) of HC-Hole.
Disulfide bond linkages are important in protein folding and play a significant role in both protein structure and functions. The number of disulfide bonds and their positions are critical attributes for biopharmaceuticals to ensure safety and efficacy.
The drug molecule has 26 cysteine residues, as shown in FIG. 3, which are cross-linked by 3 inter-chain disulfide bonds (one set of disulfide bonds between the HC-Knob and light chains, and another two sets between the HC-Knob and HC-Hole chains) and 10 intra-chain disulfide bonds. 11 disulfide bond related peptides (DS 1 to DS 11) are expected by non-reduced Lys-C/trypsin sequential digestion, because amino acid sequence 226T-K450 of CH2 and CH3 domain of HC-Knob are identical with sequence260Y-K484 of HC-Hole. Two inter-chain disulfide bonds in the hinge region between the heavy chains are observed in one peptide (DS6) upon the current digestion.
Non-reduced peptide mapping approach is often used to analyze disulfide bond linkages.
The protein samples were alkylated using N-ethylmaleimide (NEM) to protect free cysteines,  then treated by Lys-C/trypsin sequential digestion. The digested samples were divided into two aliquots. One aliquot was reduced with tris (2-carboxyethyl) phosphine (TCEP) (reduced sample, labeled as R) and the other was taken as control (non-reduced sample, labeled as NR) . The peptides were injected into an Agilent/UHPLC 1290 coupled with Thermo/Orbitrap Fusion mass spectrometer for data collection. The peptides were separated with an Agilent/Poroshell SB-C18 column, and the UV chromatograms were acquired with UV detector at the wavelength of 214 nm. The peptides were further detected by the mass spectrometer operating with full MS scan followed by tandem mass spectrometry (MS/MS) scans.
From the non-reduced samples, the corresponding disulfide bond related peptides were confirmed by comparing the measured and theoretical masses. In the reduced samples, the disulfide bond related peptides were reduced. The existence of disulfide bonds was supported by comparing the peak differences in the UV chromatograms between non-reduced and reduced samples.
The overlays of non-reduced and reduced peptide maps by Lys-C/trypsin sequential digestion are shown in Error! Reference source not found. 4. The non-reduced and reduced peptide maps were well separated. The results for the 2368S2 10720YRS NR (non-reduced) and 2368S210720YRS R (reduced) are displayed in blue and green, respectively. DS1b, DS3b, DS4b/c, DS5b, DS6b/c, DS8b, DS9b and DS11b are miss-cleaved peptides for DS1a, DS3a, DS4a, DS5a, DS6a, DS8a, DS9a and DS11a, respectively (FIG. 4) . Compared with reduced peptide maps, some UV peak differences in the non-reduced peptide maps caused by sample preparation were confirmed and the detailed information is not shown in Table 2. The UV peaks at RT 47.0 min, 51.4 min, 67.6 min, 87.3 min and 91.7 min were caused by a specific cleavages. The UV peak at RT 95.4 min was caused by over-alkylation
The detailed masses of disulfide bond related peptides for 2368S210720YRS are listed in Table 2. The peak annotation of non-reduced peptides (DS1-DS11) is shown in FIG. 4. All predicted disulfide bond related peptides were detected, and their mass differences were within 10.0 ppm compared with the theoretical ones. DS lb, DS3b, DS4b/c, DS5b, DS6b/c, DS8b, DS9b and DS11b were miss-cleaved peptides for DS1a, DS3a, DS4a, DS5a, DS6a, DS8a, DS9a and DS11a, respectively.
The disulfide bonds in the hinge region were identified in DS6a, DS6b and DS6c. All the theoretical disulfide bond related peptides were confirmed. In conclusion, the experimental data supported the predicted disulfide assignments.
Table 2. Disulfide bond assignment


HC refers to heavy chain and LC refers to light chain. The RT was reported by PMI software. DS1b, DS3b, DS4b/c, DS5b, DS6b/c, DS8b, DS9b and DS11b were miss-cleaved peptides for DS1a, DS3a, DS4a, DS5a, DS6a, DS8a, DS9a and DS11a, respectively. The difference was calculated by the following equation and the results were reported with 1 decimal place.
Example 3. Far-UV CD
Higher-order Structures
CD (Circular dichroism) is a spectroscopic technique that measures the differential absorption of left and right-handed circularly polarized light in an optically active substance. It is widely used to characterize higher order structures of protein. Chiral optical properties of proteins are originated from the asymmetric centers of their amino acid components. Proteins exhibit CD signals in the far-UV and near-UV regions characteristic of protein secondary and tertiary structures, respectively.
Far-UV CD spectrum of proteins can reveal the characteristic secondary structures, i.e. α-helix, β-sheet, random coil et al. Protein samples were diluted with ultrapure water to 0.1 mg/mL. The data collection and analysis were performed with Applied Photophysics Chirascan V1 00 CD spectrometer and Chirascan software.
The secondary structure consistency of 2368S210108Y-STD and 2368S210720YRS can be confirmed by comparing their CD spectra. The CD spectra of 2368S210108Y-STD and 2368S210720YRS in the far-UV region (190-260 nm) are shown in FIG. 5. The β-sheet and random coil were the main secondary structure compositions. The spectral similarity/structure consistency analysis was confirmed by calculating the correlation coefficient of two spectra with “Quick Compare” tool of OPUS software. No significant difference was observed in the far-UV CD between 2368S210108Y-STD and 2368S210720YRS, revealing the consistency of the secondary structure.
Example 4. Near-UV CD
Near-UV CD spectrum provides information on the protein tertiary structure. The CD spectral pattern in the 250-350 nm region is determined by the absorption, dipole orientation and the nature of the surrounding environment of the phenylalanine (250-270 nm) , tyrosine (270-290 nm) , and tryptophan (290-305 nm) , respectively. For the measurement, the protein samples were diluted with 20 mM Acetate, 8%sucrose, 0.02%PS80, pH 5.0 to 1.0 mg/mL. The data collection and analysis were performed by Applied Photophysics Chirascan V100 CD spectrometer and Chirascan software, respectively.
The CD spectra of 2368S210108Y-STD and 2368S210720YRS in the near-UV region (250-350 nm) are shown in FIG. 6. The spectral similarity/structure consistency analysis was confirmed by calculating the correlation coefficient of two spectra with “Quick Compare” tool of OPUS software. No significant difference was observed in the near-UV CD of 2368S210108Y-STD and 2368S210720YRS, revealing the consistency of the tertiary structure.
Example 5. Thermal Stability
DSC (Differential scanning calorimetry) is an experimental technique to obtain thermal transition profile of materials. It is widely used in the investigation of folding/unfolding transitions of proteins under programmed temperature increase in protein characterization. The onset (TOnset) and mid- point (Tm) temperatures of the thermal transition are commonly used as indicators of thermal stability, and the DSC thermograrn reveals the thermal transition profile.
Sample 2368S210720YRS was analyzed with MicroCal DSC from Malvem. The protein sample was diluted to 1 mg/mL with 20 mM Acetate, 8%sucrose, 0.02%PS80, pH 5.0 before analysis. 400 μL of the corresponding formulation buffer was added to a 96-well plate as the reference and 400 μtL of the protein sample was added. The samples were heated from 10 ℃ to 95 ℃ at a heating rate of 90 ℃/h in the capillary DSC system. The DSC data was analyzed and fitted with MicroCal PEAQ-DSC Software 1.51. The results are shown in FIG. 7. For 2368S210720YRS, the TOnset was determined as 60.3 ℃ and the two transition mid-point temperatures (Tm1 and Tm2) were determined as 70.0 ℃ and 74.1 ℃.
Example 6: N-GIycan Analysis
Glycosylation can play a critical role in protein stability, in vivo activity, solubility, serum half-life and immunogenicity. N-glycan analysis provides an indication on the potential safety and efficacy of therapeutic antibodies.
N-glycan analysis of the recombinant antibody was performed by releasing the N-glycans from glycoproteins with Rapid PNGase F. After enzymatic digestion, ice-cold ethanol was added into the solution to precipitate the deglycosylated protein. The supernatant was dried for 2-AB (2-Aminobenzamide) labeling. The labeled glycans were separated and detected with hydrophilic interaction liquid chromatography (HILIC) coupled to fluorescence detector (FLD) on UPLC system. Finally, each individual glycan was quantified by their relative peak area. The FLD chromatograms of N-glycans in the recombinant antibody are shown in FIG. 8A and FIG. 8B, and the detailed information of identified N-glycans is shown in Table 2. The major N-glycan forms of the recombinant antibody were G0 and G1 as depicted in structures in FIG. 9.
Table 3. Detailed information of identified N-glycans in 2368S210720YRS

Note:
1. The nomenclature of N-glycans follows GlcNAc-Hexose-Fucose-Neu5Ac-Neu5Gc. For example, 33000 represents GicNAc (3) -Hexose (3) -Fucose (0) -Neu5Ac (0) -Neu5Gc (0) .
2. a and b represent glycan isomers that show different retention. For example, G1a and G1b are isomers, thus are grouped to be G1.
3. Others indicate sum of un-identified glycans.
Example 7: Formulation
The recombinant antibody was dialyzed into eleven formulation buffers at a concentration of 20.0mg/mL represented in Table 4 below. A sample of each formulation was run on HPLC to determine the precent aggregate in solution. The recombinant antibody was more stable in acetate and histidine buffer that citrate or phosphate buffer (PB) . The recombinant antibody was found to be more stable in low-pH buffers.
Table 4. Formulation Buffer
Example 8: Trial to evaluate the bispecific antibody targeting CD38 and ICAM1
This is a non-randomized, sequential assignment open label phase 1 interventional trial that will test the safety, tolerability, and pharmacokinetics of an isolated recombinant antibody targeting CD38 and ICAM1 (an afucosylated humanized Fc-modified immunoglobulin G1 bispecific antibody, hereinafter referred to as “composition” which is described herein) in trial participants (referred to as “subject” or “subjects” hereinafter) with relapsed or refractory multiple myeloma, lymphoma, or solid tumors.
Inclusion criteria: a random population of 110 adult participants 18 years and older are enrolled  in the trial following a histologic diagnosis of a relapsed or refractory solid tumor, refractory myeloma, or lymphoma with measurable or evaluable disease. Subjects must have progressed following all therapies of known, potential clinical benefit or for whom treatments of known clinical benefit are contraindicated. Subjects must have adequate kidney, liver, and hematologic function with an Eastern Cooperative Oncology Group (ECOG) performance status 0 to 2. The trial is conducted in two parts:
Part 1: Dose escalation: is conducted to evaluate increasing doses of the composition to identify the maximum tolerated dose (MTD) or recommended Phase 2 dose (RP2D) . For the dose escalation trial, subjects or patient population with a diagnosis ora relapsed or refractory multiple myeloma, lymphoma or solid tumors, with measurable or evaluable disease enrolled in the trial receive the composition administered as an intravenous infusion weekly for 6 weeks then every 2 weeks. The first subject enrolled receives the lowest dose of the composition. Once the lowest dose is shown to be safe, an additional subject is enrolled at the next higher dose. Subjects continue to be enrolled into either single or multiple patient groups receiving increasing doses until the MTD or RP2D is reached.
Part 2: Dose expansion: Following completion of the dose escalation of the trial (part 1 above) and determination of the maximum tolerated dose or recommended phase 2 dose, subjects (subjects or patient population described above) who have relapsed myeloma and lymphoma are enrolled in the trial and treated with the composition at the MTD or RP2D obtained in trial part 1.
Primary Outcome Measures:
1. Occurrence of general toxicity: the incidence of treatment-emergent serious adverse events such as toxicity and change from baseline in safety parameters is monitored for a period of 30 months.
2. Occurrence of dose limiting toxicity: the incidence of dose limiting toxicity is monitored during the first cycle (cycle 1) of the dose escalation for a period covering the first 21 days of administering the composition (dosing period) .
Secondary Outcome Measures:
1. The serum concentrations of the composition are measured to determine the changes in serum levels from levels seen at baseline. Measurements are conducted for the duration of the trial for a period of 30 months.
2. The serum levels of the composition (antidrug) and levels of neutralizing antibodies are measured to determine the changes in levels of the antidrug and neutralizing antibodies since baseline. Measurements are conducted for the duration of the trial for a period of 30 months.
3. Measure objective response, which can be assessed by International Myeloma Working Group (IMWG) for multiple myeloma, the Lugano criteria for lymphoma or RECIST 1.1 for solid tumors. Measurements are conducted for the duration of the trial for a period of 30 months.
4. Measure the best response, which can be assessed by IMWG response criteria for multiple myeloma, the Lugano criteria for lymphoma or RECIST 1.1 for solid tumors. Measurements are conducted for the duration of the trial for a period of 30 months.
5. Measure time to response and duration of response, which can be assessed by IMWG for multiple myeloma or the Lugano criteria for lymphoma or RECIST 1.1 for solid tumors. Measurements  are conducted for the duration of the trial for a period of 30 months.
6. Measure progress-free survival, which can be assessed by IMWG for multiple myeloma or the Lugano criteria for lymphoma or RECIST 1.1 for solid tumors. Measurements are conducted for the duration of the trial for a period of 30 months.
Other Outcome Measures
1. Measure tumor expression, which can be evaluated by measuring ICAM1 and CD38 expression with clinical outcomes. Measurements are conducted for the duration of the trial for a period of 30 months.
2. Measure immunoglobulins, which can be assessed by measuring the quantitative immunoglobulins with clinical outcomes. Measurements are conducted for the duration of the trial for a period of 30 months.
Inclusion Criteria:
All adults 18 years and older
Histologic diagnosis of a refractory solid tumor, refractory myeloma, or lymphoma with measurable or evaluable disease
Patients must have progressed following all therapies of known, potential clinical benefit, or for whom treatments of known clinical benefit are contraindicated.
Adequate kidney, liver, and hematologic function
Eastern Cooperative Ontology Group (ECOG) performance status 0 to 2
Exclusion criteria
Active brain metastases and history of leptomeningeal metastases.
Myeloma patients with plasmacytoma as only measurable disease
Non-secretory myeloma
Patients with advanced metastatic, symptomatic, visceral spread who are at risk of life-threatening complications
Active or chronic, uncontrolled bacterial, viral, or fungal infection (s)
Abnormal ECG
Has clinically significant cardiovascular disease
Additional active malignancy that may confound the assessment of the study endpoints
Pregnancy or lactation
Known seropositivity for HIV (human immunodeficiency virus) or active hepatitis B or hepatitis C

Claims (112)

  1. An isolated recombinant antibody that targets CD38 and ICAM1 comprising a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3, and wherein the isolated recombinant antibody has at least one of the following characteristics:
    (a) at least one disulfide bond formed by a pair of cysteine residues;
    (b) a far UV circular dichroism peak at a wavelength between 195 nm and 210 nm when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0;
    (c) a near UV circular diehroism peak at a wavelength between 270 nm and 300nm when the isolated recombinant 3 antibody is formulated at a concentration of 1.0 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0;
    (d) an onset melting temperature (T0) between 58 ℃ to 62 ℃, a transition midpoint temperature 1 (Tm1) between 68 ℃ to 71 ℃, a transition midpoint temperature 2 (Tm2) between 72 ℃ to 75 ℃, when the isolated recombinant antibody is formulated at a concentration of 1 mg/mL in 20 mM acetate, 8%sucrose, 0.02%polysorbate 80, pH 5.0; and
    (e) at least one N-glycan moiety.
  2. The isolated recombinant antibody of claim 1, wherein the LC comprises at least 85%sequence identity to SEQ ID NO: 1.
  3. The isolated recombinant antibody of claim 1, wherein the LC comprises at least 90%sequence identity to SEQ ID NO: 1.
  4. The isolated recombinant antibody of claim 1, wherein the LC comprises at least 95%sequence identity to SEQ ID NO: 1.
  5. The isolated recombinant antibody of claim 1, wherein the LC comprises at least 99%sequence identity to SEQ ID NO: 1.
  6. The isolated recombinant antibody of claim 1, wherein the LC comprises the amino acid sequence according to SEQ ID NO: 1.
  7. The isolated recombinant antibody of claim 1, wherein the HC-Knob comprises at least 85%sequence identity to SEQ ID NO: 2.
  8. The isolated recombinant antibody of claim 1, wherein the HC-Knob comprises at least 90%sequence identity to SEQ ID NO: 2.
  9. The isolated recombinant antibody of claim 1, wherein the HC-Knob comprises at least 95%sequence identity to SEQ ID NO: 2.
  10. The isolated recombinant antibody of claim 1, wherein the HC-Knob comprises at least 99%sequence identity to SEQ ID NO: 2.
  11. The isolated recombinant antibody of claim 1, wherein the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2.
  12. The isolated recombinant antibody of claim 1, wherein the HC-Hole comprises at least 85%sequence identity to SEQ ID NO: 3.
  13. The isolated recombinant antibody of claim 1, wherein the HC-Hole comprises at least 90%sequence identity to SEQ ID NO: 3.
  14. The isolated recombinant antibody of claim 1, wherein the HC-Hole comprises at least 95%sequence identity to SEQ ID NO: 3.
  15. The isolated recombinant antibody of claim 1, wherein the HC-Hole comprises at least 99%sequence identity to SEQ ID NO: 3.
  16. The isolated recombinant antibody of claim 1, wherein the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  17. The isolated recombinant antibody of claim 1, wherein the LC comprises at least 99%sequence identity to SEQ ID NO: 1, the HC-Knob comprises at least 99%sequence identity to SEQ ID NO:2, HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  18. The isolated recombinant antibody of claim 1, wherein the LC comprises the amino acid sequence according to SEQ ID NO: 1, the HC-Knob comprises the amino acid sequence according to SEQ ID NO: 2, the HC-Hole comprises the amino acid sequence according to SEQ ID NO: 3.
  19. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least two of the characteristics.
  20. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least three of the characteristics.
  21. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least four of the characteristics.
  22. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least five of the characteristics.
  23. The isolated recombinant antibody of claim 1, wherein the at least one disulfide bond is an intrachain disulfide bond.
  24. The isolated recombinant antibody of claim 1, wherein the at least one disulfide bond is an interchain disulfide bond.
  25. The isolated recombinant antibody of claim 1, wherein the interchain disulfide bond is between the HC-Knob and the HC-Hole.
  26. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least two disulfide bonds formed by pairs of cysteine residues.
  27. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least three disulfide bonds formed by pairs of cysteine residues.
  28. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least four disulfide bonds formed by pairs of cysteine residues.
  29. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least five disulfide bonds formed by pairs of cysteine residues.
  30. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least six disulfide bonds formed by pairs of cysteine residues.
  31. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least seven disulfide bonds formed by pairs of cysteine residues.
  32. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least eight disulfide bonds formed by pairs of cysteine residues.
  33. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least nine disulfide bonds formed by pairs of cysteine residues.
  34. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least ten disulfide bonds formed by pairs of cysteine residues.
  35. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least eleven disulfide bonds formed by pairs of cysteine residues.
  36. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least twelve disulfide bonds formed by pairs of cysteine residues.
  37. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least thirteen disulfide bonds formed by pairs of cysteine residues.
  38. The isolated recombinant antibody of claim 1, wherein the isolated recombinant antibody comprises at least fourteen disulfide bonds formed by pairs of eysteine residues.
  39. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 23 and Cysteine 88 of SEQ ID NO: 1.
  40. The isolated recombinant antibody of claim 26 wherein the pair of cysteine residues comprises Cysteine 133 and Cysteine 197 of SEQ ID NO: 1.
  41. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1.
  42. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2.
  43. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 2.
  44. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3.
  45. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3.
  46. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3.
  47. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 3.
  48. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 3.
  49. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3.
  50. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3.
  51. The isolated recombinant antibody of claim 26, wherein the pair of cysteine residues comprises Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  52. The isolated recombinant antibody of claim 1, wherein the at least one disulfide bond formed by a pair of cysteine residues is selected from
    Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 ofSEQ ID NO: 1;
    Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
    Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
    Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
    Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
    Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
    Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
    Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 ofSEQ ID NO: 3; and
    Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  53. The isolated recombinant antibody of claim 31, wherein the at least seven disulfide bonds formed by a pair of cysteine residues is selected from
    Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
    Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
    Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
    Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
    Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
    Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
    Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
    Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
    Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  54. The isolated recombinant antibody of claim 34, wherein the at least ten disulfide bonds formed by a pair of cysteine residues is selected from
    Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
    Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
    Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
    Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
    Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
    Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
    Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
    Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
    Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  55. The isolated recombinant antibody of claim 36, wherein the at least 12 disulfide bonds formed by a pair of cysteine residues is selected from
    Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
    Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
    Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
    Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
    Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
    Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 ofSEQ ID NO: 2;
    Cysteine 23 ofSEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
    Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
    Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  56. The isolated recombinant antibody of claim 37, wherein the at least 13 disulfide bonds formed by a pair of cysteine residues is selected from
    Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
    Cysteine 137 ofSEQ ID NO: 1 and Cysteine 197 ofSEQ ID NO: 1;
    Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
    Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
    Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
    Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
    Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
    Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
    Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  57. The isolated recombinant antibody of claim 38, wherein the 14 disulfide bonds are formed by a pair of cysteine residues selected from
    Cysteine 23 of SEQ ID NO: 1 and Cysteine 88 of SEQ ID NO: 1;
    Cysteine 137 of SEQ ID NO: 1 and Cysteine 197 of SEQ ID NO: 1;
    Cysteine 223 of SEQ ID NO: 2 and Cysteine 217 of SEQ ID NO: 1;
    Cysteine 22 of SEQ ID NO: 2 and Cysteine 95 of SEQ ID NO: 2;
    Cysteine 147 of SEQ ID NO: 2 and Cysteine 203 of SEQ ID NO: 2;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 229 of SEQ ID NO: 2 and Cysteine 266 of SEQ ID NO: 3;
    Cysteine 232 of SEQ ID NO: 2 and Cysteine 263 of SEQ ID NO: 3;
    Cysteine 264 of SEQ ID NO: 2 and Cysteine 324 of SEQ ID NO: 2;
    Cysteine 370 of SEQ ID NO: 2 and Cysteine 428 of SEQ ID NO: 2;
    Cysteine 23 of SEQ ID NO: 3 and Cysteine 90 of SEQ ID NO: 3;
    Cysteine 147 of SEQ ID NO: 3 and Cysteine 222 of SEQ ID NO: 3; and
    Cysteine 404 of SEQ ID NO: 3 and Cysteine 462 of SEQ ID NO: 3.
  58. The isolated recombinant antibody of claim 1, wherein the far UV circular dichroism peak is at a wavelength between 200 nm and 205 nm.
  59. The isolated recombinant antibody of claim 1, wherein the near UV circular dichroism peak is at a wavelength between 270 nm and 290 nm.
  60. The isolated recombinant antibody of claim 1, wherein the near UV circular dichroism peak is at a wavelength between 280 nm and 290 nm.
  61. The isolated recombinant antibody of claim 1, wherein T0 is between 59 ℃ to 61 ℃, Tm1 is between 69 ℃ to 71 ℃, and Tm2 is between 71 ℃ to 74 ℃.
  62. The isolated recombinant antibody of claim 1, wherein the T0 is 60.3 ℃, Tm1 is 70.0 ℃, and Tm2 is 74.1 ℃.
  63. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises N-acetylglucosamine (GlcNAc) , hexose, fucose, N-Acetylneuraminic acid (Neu5Ac) , or N-Glycolylneuraminic acid (Neu5Gc) .
  64. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises GlcNAc, hexose, or Neu5Gc.
  65. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises GlcNAc and hexose.
  66. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises GlcNAc, hexose, and Neu5Gc.
  67. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises at least two GlcNAc moieties and at least two hexose moieties.
  68. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises at least three GlcNAc moieties and at least three hexose moieties.
  69. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and three hexose moieties.
  70. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and three hexose moieties.
  71. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and four hexose moieties.
  72. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises two GlcNAc moieties and five hexose moieties.
  73. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises five GlcNAc moieties and three hexose moieties.
  74. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and four hexose moieties.
  75. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises three GlcNAc moieties and five hexose moieties.
  76. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises five GlcNAc moieties and four hexose moieties.
  77. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises four GlcNAc moieties and five hexose moieties.
  78. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises four GlcNAc moieties, five hexose moieties, and one Neu5Ac moiety.
  79. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety comprises two GlcNAc moieties and eight hexose moieties.
  80. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety is at Asparagine 300 of SEQ ID NO: 2.
  81. The isolated recombinant antibody of claim 1, wherein the at least one N-glycan moiety is at Asparagine 334 of SEQ ID NO: 3.
  82. A plurality of isolated recombinant antibodies that target CD38 and ICAM1 wherein the isolated recombinant antibodies comprise a light chain (LC) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 1, a heavy chain-knob (HC-Knob) amino acid sequence that has at least 80 %sequence identity to SEQ ID NO: 2, a heavy chain hole (HC-Hole) amino acid sequence that has at least 80%sequence identity to SEQ ID NO: 3 wherein the plurality comprises greater than 90%monomer of the isolated recombinant antibodies.
  83. The plurality of isolated recombinant antibodies of claim 82, comprising greater than 95%monomer.
  84. The plurality of isolated recombinant antibodies of claim 82, comprising greater than 99%monomer.
  85. The plurality of isolated recombinant antibodies of claim 82, wherein the concentration of the isolated recombinant antibodies is greater than or equal to 1.0 mg/mL.
  86. The plurality of isolated recombinant antibodies of claim 82, wherein the concentration of the isolated recombinant antibodies is at least 2.0 mg/mL.
  87. The plurality of isolated recombinant antibodies of claim 82, wherein the concentration of the isolated recombinant antibodies is at least 5.0 mg/mL.
  88. The plurality of isolated recombinant antibodies of claim 82, wherein the concentration of the isolated recombinant antibodies is at least 10.0 mg/mL.
  89. The plurality of isolated recombinant antibodies of claim 82, wherein the concentration of the isolated recombinant antibodies is at least 15.0 mg/mL.
  90. The plurality of isolated recombinant antibodies of claim 82, wherein the concentration of the isolated recombinant antibodies is at least 20.0 mg/mL.
  91. The plurality of isolated recombinant antibodies of claim 82, wherein the plurality is in a buffered solution having a pH less than or equal to 5.5.
  92. The plurality of isolated recombinant antibodies of claim 82, wherein the buffered solution comprises one or more of acetate, phosphate, or histidine.
  93. The plurality of isolated recombinant antibodies of claim 82, wherein the buffered solution comprises acetate at a concentration greater than 15mM.
  94. The plurality of isolated recombinant antibodies of claim 82, wherein the buffered solution comprises sucrose.
  95. The plurality of isolated recombinant antibodies of claim 94, wherein the sucrose is at a concentration greater than or equal to 5%w/v.
  96. The plurality of isolated recombinant antibodies of claim 82, wherein the buffered solution comprises polysorbate 80.
  97. The plurality of isolated recombinant antibodies of claim 96, wherein the polysorbate 80 is at a concentration greater than or equal to 0.01%w/v.
  98. The plurality of isolated recombinant antibodies of claim 82, comprising greater than 90%monomer at a concentration of greater than or equal to 20.0mg/mL in 20 mM acetate, 8% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH of 5.0.
  99. A method of treating a cancer in a subject in need thereof comprising administering to the subject the isolated recombinant antibody of claim 1, or the plurality of isolated recombinant antibodies of claims 81-97, wherein the cancer comprises a cell that expresses CD38 and ICAM1.
  100. The method of claim 99, wherein the cancer comprises a solid tumor or a hematological malignancy.
  101. The method of claim 99, wherein the cancer comprises the hematological malignancy.
  102. The method of claim 101, wherein the hematological malignancy is multiple myeloma, lymphoma, or Burkitt lymphoma.
  103. The method of claim 99, wherein the cancer is a lung cancer or a prostate cancer.
  104. The method of claim 100, wherein the solid tumor comprises a refractory solid tumor.
  105. The method of claim 100, wherein the solid tumor comprises a relapsed solid tumor.
  106. The method of claim 102, wherein the multiple myeloma comprises refractory multiple myeloma.
  107. The method of claim 102, wherein the multiple myeloma comprises relapsed multiple myeloma.
  108. The method of claim 102, wherein the lymphoma comprises refractory lymphoma.
  109. The method of claim 102, wherein the lymphoma comprises relapsed lymphoma.
  110. The method of claim 102, wherein the subject has received a diagnosis of a relapsed or refractory sol id tumor.
  111. The method of claim 102, wherein the subject has received a diagnosis of relapsed or refractory multiple myeloma.
  112. The method of claim 102, wherein the subject has received a diagnosis of relapsed or refractory lymphoma.
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