WO2023143478A1 - Novel anti-cd4 and anti-pd-l1 bispecific antibodies - Google Patents
Novel anti-cd4 and anti-pd-l1 bispecific antibodies Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
- C07K16/4258—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig
- C07K16/4275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig against anti-receptor Ig against anti-CD4 Ig
-
- 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
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
-
- 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
- the present disclosure relates generally to the fields of medicine, oncology, and immunology. More particular, the disclosure relates to bispecific antibodies that bind to CD4 and PD-L1 and to methods of using such bispecific antibodies.
- CD4 is a monomeric type I transmembrane glycoprotein comprising four extracellular Ig-like domains, a hydrophobic transmembrane domain, and a highly basic cytoplasmic tail (Glatzova D, et al., Front Immunol (2019) 10: 618) .
- the N-terminal Ig-like domains of CD4 interact with the ⁇ 2 and ⁇ 2 domains of MHC class II molecules to assemble TCR–pMHC–CD4 ternary complex, and the resulting close proximity between the TCR and CD4 allows the tyrosine kinase Lck bound to the cytoplasmic tail of CD4 to phosphorylate tyrosine residues of immunoreceptor tyrosine activation motifs (ITAMs) on the cytoplasmic domains of CD3 to amplify the signal generated by the TCR (Rudd CE, et al., Proc Natl Acad Sci USA (1988) 85: 5190; Barber EK, et al., Proc Natl Acad Sci USA (1989) 86: 3277 and Li QJ, et al., Nat Immunol (2004) 5: 791) .
- ITAMs immunoreceptor tyrosine activation motifs
- CD4 is expressed in a large proportion of thymocytes (80–90%) and over 50%of the peripheral blood T-cells (Nakamura K, et al., Mol Immunol (2003) 39: 909) . CD4 is also expressed to a minor extent on macrophages, monocytes, dendritic cells and Langerhans cells.
- CD4 plays an important role not only in the differentiation of thymocytes and the regulation of T-lymphocyte/B-lymphocyte adhesion, but also in MHC class II-restricted T cell activation (Gaubin M, et al., Eur J Clin Chem Clin Biochem (1996) 34: 723; Artyomov MN, et al., Proc Natl Acad Sci USA (2010) 107: 16916; Yili L, et al., Front Immunol (2013) 4: 206) .
- CD4 expressing cells such as regulatory T cells (Tregs) have been shown to suppress tumor-induced immunity, thereby compromising the therapeutic efficacy of various cancer therapies (Baba J, et al., Blood (2012) 120: 2417; Togashi Y, et al., Nature Reviews Clinical Oncology (2019) 16: 356) .
- Programmed death-ligand 1 is a type 1 transmembrane protein that plays a major role in suppressing the adaptive immune response. Normally the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals. In turn, clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated.
- the binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM) . This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells.
- SHP-1 or SHP-2 phosphatases
- the present disclosure provides anti-CD4 and anti-PD-L1 bispecific antibodies and antigen-binding fragment thereof, amino acid and nucleotide sequences thereof, and uses thereof.
- the present disclosure provides an anti-CD4 and anti-PD-L1 bispecific antibody or an antigen-binding fragment thereof.
- the anti-CD4 and anti-PD-L1 bispecific antibody or an antigen-binding fragment comprises a CD4 binding domain and a PD-L1 binding domain.
- the CD4 binding domain comprise (i) a heavy chain (HC) variable region (VH) comprising a HCDR1 comprising the sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the sequence set forth in SEQ ID NO:2, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 3, and variants thereof wherein one or more of the HC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof, and (ii) a light chain (LC) variable region (VL) comprising a LCDR1 comprising the sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the sequence set forth in SEQ ID NO: 6, and variants thereof wherein one or more of the LC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof.
- HC heavy chain
- VH heavy chain variable region
- the PD-L1 binding domain comprises (i) a VH comprising a HCDR1 comprising the sequence set forth in SEQ ID NO: 11, a HCDR2 comprising the sequence set forth in SEQ ID NO: 12, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 13, and variants thereof wherein one or more of the HC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof, and (ii) a VL comprising a LCDR1 comprising the sequence set forth in SEQ ID NO: 14, a LCDR2 comprising the sequence set forth in SEQ ID NO: 15, and a LCDR3 comprising the sequence set forth in SEQ ID NO: 16, and variants thereof wherein one or more of the LC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof.
- the VH of the CD4 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 7
- the VL of the CD4 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 8.
- the VH of the PD-L1 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 17, and the VL of the PD-L1 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 18.
- the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region.
- the heavy chain constant region comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4.
- the heavy chain constant region comprises a constant region of human IgG1.
- the IgG1 comprises one or more mutations that can confer increased CDC or ADCC relative to wild-type constant region.
- the one or more mutations is selected from the group consisting of S239D, I332E, H268F, S324T S236A, G236A, P247I, A339 (D/Q) , D280H, K290S, S298 (D/V) , F243L, R292P, Y300L, P396L, V305I, K290 (E/N) , S298G, T299A, K326E, E382V, M428I, S298A, K326A, E333A, K334A, S298A, E333A, and K334A, according to EU numbering.
- the one or more mutations comprise a combination of S298A, E333A, and K334A, according to EU numbering.
- the PD-L1 binding domain is a scFv (single chain fragment variable) domain.
- the scFv domain is linked to a heavy chain constant region.
- the scFv domain is linked to a light chain constant region.
- the anti-CD4 and anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 21; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 10.
- the anti-CD4 and anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 9; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 22.
- the isolated antibody is a murine, a rodent, a rabbit, a chimeric, a humanized, or a human antibody.
- the antigen-binding fragment is a recombinant ScFv (single chain fragment variable) antibody, a Fab fragment, a F (ab’) 2 fragment, or a Fv fragment.
- the isolated antibody or an antigen-binding fragment thereof disclosed herein is linked to one or more conjugate moieties.
- the conjugate moiety is an immune-modulatory agent, an anti-tumor drug, a clearance-modifying agent, a toxin, a detectable label, an RNA, a DNA, a cytokine, or purification moiety.
- the present disclosure provides a pharmaceutical composition
- a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.
- the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof disclosed herein.
- the present disclosure provides a vector comprising the polynucleotide encoding the antibody or antigen-binding fragment thereof disclosed herein.
- the present disclosure provides a host cell comprising the vector disclosed herein.
- the present disclosure provides a method of producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell disclosed herein under the condition at which the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.
- the present disclosure provides a method of treating or ameliorating the effect of a cancer in a subject.
- the method comprises administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof disclosed herein, or the pharmaceutical composition disclosed herein.
- the cancer is selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer.
- the subject is human.
- the antibody or an antigen-binding fragment thereof is administered intravenously, intra-arterially, intra-tumorally, intra-muscularly, or subcutaneously.
- the present disclosure provides use of the antibody or antigen-binding fragment thereof disclosed herein in the manufacture of a medication for treating cancer in a subject.
- FIG. 1 shows the configuration of an exemplary bispecific antibody.
- FIG. 2 shows the configuration of an exemplary bispecific antibody.
- FIG. 3 shows binding of anti-CD4/anti-PD-L1 bispecific antibody with human CD4.
- FIG. 4 shows binding of anti-CD4/anti-PD-L1 bispecific antibody with human PD-L1.
- FIG. 5 shows the secretion of interleukin 2 (IL-2) induced by anti-CD4/anti-PD-L1 bispecific antibody.
- IL-2 interleukin 2
- FIG. 6 shows the secretion of interferon ⁇ (IFN- ⁇ ) induced by anti-CD4/anti-PD-L1 bispecific antibody.
- FIG. 7 shows antibody-dependent cell-mediated cytotoxicity (ADCC) effect of anti-CD4/anti-PD-L1 bispecific antibody.
- antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multi-specific antibody, or bispecific antibody that binds to a specific antigen.
- a native intact antibody comprises two heavy (H) chains and two light (L) chains.
- Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable domain (V H ) and a constant region including a first, second, and third constant domain (C H1 , C H2 , C H3 , respectively) ; mammalian light chains are classified as ⁇ or ⁇ , while each light chain consists of a variable domain (V L ) and a constant domain (C L ) .
- a typical IgG antibody has a “Y”shape, with the stem of the Y typically consisting of the second and third constant domains of two heavy chains bound together via disulfide bonding.
- Each arm of the Y includes the variable domain and first constant domain of a single heavy chain bound to the variable and constant domains of a single light chain.
- the variable domains of the light and heavy chains are responsible for antigen binding.
- the variable domains in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) .
- CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol.
- the three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
- FRs framework regions
- the constant domains of the heavy and light chains are not involved in antigen-binding but exhibit various effector functions.
- Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
- the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively.
- IgG1 gamma1 heavy chain
- IgG2 gamma2 heavy chain
- IgG3 gamma3 heavy chain
- IgG4 gamma4 heavy chain
- IgA1 alpha1 heavy chain
- IgA2 alpha2 heavy chain
- an antigen refers to a substance capable of inducing adaptive immune responses.
- an antigen is a substance specifically bound by antibodies or T lymphocyte antigen receptors.
- Antigens are usually proteins and polysaccharides, less frequently also lipids. Suitable antigens include without limitation parts of bacteria (coats, capsules, cell walls, flagella, fimbrai, and toxins) , viruses, and other microorganisms.
- Antigens also include tumor antigens, e.g., antigens generated by mutations in tumors.
- antigens also include immunogens and haptens.
- antigen-binding fragment refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure.
- antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2 , an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2 , a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific antibody, a multi-specific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.
- a “Fab fragment” comprises one light chain and the C H 1 and variable domains of one heavy chain.
- the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
- a “Fab′fragment” comprises one light chain and a portion of one heavy chain that contains the V H domain and the C H 1 domain and also the region between the C H 1 and C H 2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F (ab′) 2 molecule.
- a “F (ab′) 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the C H 1 and C H 2 domains, such that an interchain disulfide bond is formed between the two heavy chains.
- a F (ab′) 2 fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the two heavy chains.
- Fv with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site.
- An Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain.
- Single-chain Fv antibody or “scFv” refers to an engineered antibody consisting of a light chain variable domain and a heavy chain variable domain connected to one another directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA (1988) 85: 5879) .
- An “Fc” region comprises two heavy chain fragments comprising the C H 2 and C H 3 domains of an antibody.
- the two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C H 3 domains.
- the Fc region of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) , and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.
- Single-chain Fv-Fc antibody or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
- a “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable domain of a single light chain and the variable domain of a single heavy chain is a disulfide bond.
- a “ (dsFv) 2 ” or “ (dsFv-dsFv') ” comprises three peptide chains: two V H domains linked by a peptide linker (e.g., a long flexible linker) and bound to two V L domains, respectively, via disulfide bridges.
- dsFv-dsFv' is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.
- “Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two V H domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) .
- Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) .
- variable domain of a heavy chain antibody represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. (2007) 21: 3490-8) .
- a “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
- “Diabodies” or “dAbs” include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a V H domain connected to a V L domain in the same polypeptide chain (V H -V L or V L -V H ) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A.Jul 15; 90 (14) : 6444-8 (1993) ; EP404097; WO93/11161) .
- the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites.
- the antigen–binding sites may target the same or different antigens (or epitopes) .
- a “bispecific ds diabody” is a diabody target two different antigens (or epitopes) .
- an “scFv dimer” is divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) that can be engineered by linking two scFvs.
- a bivalent diabody or bivalent scFv (BsFv, di-scFvs, bi-scFvs) comprising V H -V L (linked by a peptide linker) dimerized with another V H -V L moiety such that V H 's of one moiety coordinate with the V L 's of the other moiety and form two binding sites which can target the same antigens (or epitopes) or different antigens (or epitopes) .
- an “scFv dimer” is a bispecific diabody comprising V H1 -V L2 (linked by a peptide linker) associated with V L1 -V H2 (also linked by a peptide linker) such that V H1 and V L1 coordinate and V H2 and V L2 coordinate and each coordinated pair has a different antigen specificity.
- a “domain antibody” refers to an antibody fragment containing only the variable domain of a heavy chain or the variable domain of a light chain.
- two or more V H domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody.
- the two V H domains of a bivalent domain antibody may target the same or different antigens.
- a “bispecific” antibody refers to an artificial antibody which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes.
- the two epitopes may present on the same antigen, or they may present on two different antigens.
- Cancer refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia.
- solid tumor refers to a solid mass of neoplastic and/or malignant cells.
- cancer or tumors include hematological malignancies, oral carcinomas (for example of the lip, tongue or pharynx) , digestive organs (for example esophagus, stomach, small intestine, colon, large intestine, or rectum) , peritoneum, liver and biliary passages, pancreas, respiratory system such as larynx or lung (small cell and non-small cell) , bone, connective tissue, skin (e.g., melanoma) , breast, reproductive organs (fallopian tube, uterus, cervix, testicles, ovary, or prostate) , urinary tract (e.g., bladder or kidney) , brain and endocrine glands such as the thyroid.
- oral carcinomas for example of the lip, tongue or pharynx
- digestive organs for example esophagus, stomach, small intestine, colon, large intestine, or rectum
- peritoneum liver and biliary passages
- the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular cancer, and colorectal cancer. In certain embodiments, the cancer is selected from a lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma.
- chimeric means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species.
- a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse or rabbit.
- the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
- the term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen.
- the antibodies or antigen-binding fragments provided herein specifically bind to human CD4 or PD-L1 with a binding affinity (K D ) of ⁇ 10 -6 M (e.g., ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 -8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 - 9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M) .
- K D binding affinity
- K D used herein refers to the ratio of the dissociation rate to the association rate (k off /k on ) , which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method.
- the K D value can be appropriately determined by using flow cytometry.
- the ability to “block binding” or to “compete for the same epitope” as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human CD4 and an anti-CD4 antibody) to any detectable degree.
- an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.
- a given antibody binds to the same epitope as the antibody of present disclosure by ascertaining whether the former prevents the latter from binding to a CD4 antigen polypeptide. If the given antibody competes with the antibody of present disclosure, as shown by a decrease in binding by the antibody of present disclosure to the CD4 antigen polypeptide, then the two antibodies bind to the same, or a closely related, epitope. Or if the binding of a given antibody to the CD4 antigen polypeptide was inhibited by the antibody of present disclosure, then the two antibodies bind to the same, or a closely related, epitope.
- CD4 refers to CD4 derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats) .
- mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats) .
- Exemplary sequence of human CD4 includes GenBank SEQ Reference No. NP_000607, NP_001181943, NP_001181944, NP_001181945, and NP_001181946.
- CD4 as used herein is intended to encompass any form of human CD4, for example, 1) native unprocessed CD4 molecule, “full-length” CD4 chain or naturally occurring variants of CD4, including, for example, splice variants or allelic variants; 2) any form of CD4 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of CD4 subunit generated through recombinant method.
- a fragment e.g., a truncated form, an extracellular/transmembrane domain
- a modified form e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form
- PD-L1 refers to PD-L1 derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats) .
- mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats) .
- Exemplary sequence of human PD-L1 includes GenBank SEQ Reference No. NP_001254635, NP_001300958, and NP_054862.
- PD-L1 as used herein is intended to encompass any form of human PD-L1, for example, 1) native unprocessed PD-L1 molecule, “full-length” PD-L1 chain or naturally occurring variants of PD-L1, including, for example, splice variants or allelic variants; 2) any form of PD-L1 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of PD-L1 subunit generated through recombinant method.
- a fragment e.g., a truncated form, an extracellular/transmembrane domain
- a modified form e.g. a mutated form, a glycosylated/PEG
- anti-CD4 anti-PD-L1 bispecific antibody refers to an antibody that is capable of simultaneously specifically binding to CD4 (e.g., human CD4) and PD-L1 (e.g., human PD-L1) .
- a “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties.
- conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g., Asp, Glu) , among amino acids with basic side chains (e.g., His, Lys, and Arg) , or among residues with aromatic side chains (e.g., Trp, Tyr, and Phe) .
- conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
- effector functions refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor.
- exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis.
- CDC complement dependent cytotoxicity
- ADCC antibody-dependent cell-mediated cytotoxicity
- epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding fragment may be considered to bind the same/closely related epitope as the reference antibody.
- homologue and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequence when optimally aligned.
- host cell refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
- humanized means that the antibody or antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
- an “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both.
- a polynucleotide or a polypeptide naturally present in a living animal is not “isolated, ” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state.
- An “isolated nucleic acid sequence” refers to the sequence of an isolated nucleic acid molecule.
- an “isolated antibody or antigen-binding fragment thereof” refers to the antibody or antigen-binding fragments having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) , or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC) .
- electrophoretic methods such as SDS-PAGE, isoelectric focusing, capillary electrophoresis
- chromatographic methods such as ion exchange chromatography or reverse phase HPLC
- leader peptide refers to a peptide having a length of about 5-30 amino acids that is present at the N-terminus of newly synthesized proteins that form part of the secretory pathway.
- Proteins of the secretory pathway include, but are not limited to proteins that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes) , are secreted from the cell, or are inserted into a cellular membrane.
- the leader peptide forms part of the transmembrane domain of a protein.
- link refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and/or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.
- operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
- a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell.
- a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence.
- the promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
- Percent (%) sequence identity with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S. F.
- polynucleotide or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers.
- the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
- Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2′, 3′-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
- polypeptide or “protein” means a string of at least two amino acids linked to one another by peptide bonds. Polypeptides and proteins may include moieties in addition to amino acids (e.g., may be glycosylated) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “polypeptide” or “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a polypeptide or protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers.
- pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
- the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) .
- a human includes pre-and post-natal forms.
- a subject is a human being.
- a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
- the term “subject” is used herein interchangeably with “individual” or “patient. ”
- a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
- a therapeutically effective amount refers to the dosage or concentration of a drug effective to treat a disease or condition.
- a therapeutically effective amount is the dosage or concentration of the monoclonal antibody or antigen-binding fragment thereof capable of reducing the tumor volume, eradicating all or part of a tumor, inhibiting or slowing tumor growth or cancer cell infiltration into other organs, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting or slowing tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.
- Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
- vector refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein.
- a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
- vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
- a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication.
- a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
- a vector can be an expression vector or a cloning vector.
- the present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
- promoter e.g., SV40, CMV, EF-1 ⁇
- vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBA
- RTM. pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
- the present disclosure in one aspect provides an anti-CD4 anti-PD-L1 bispecific antibody and antigen-binding fragment thereof that has a high binding affinity to both human CD4 and human PD-L1.
- the bispecific antibody or antigen-binding fragment when bound to PD-L1, can specifically interfere with, block or reduce the interaction between PD-L1 and its receptor PD-1.
- the bispecific antibodies or antigen-binding fragments, when bound to CD4 induces CDC or ADCC.
- Binding affinity of the antibody and antigen-binding fragment provided herein can be represented by K D value, which represents the ratio of dissociation rate to association rate (k off /k on ) when the binding between the antigen and antigen-binding molecule reaches equilibrium.
- the antigen-binding affinity e.g., K D
- K D can be appropriately determined using suitable methods known in the art, including, for example, bio-layer interferometry.
- Binding of the antibodies to human CD4 or PD-L1 can also be represented by “half maximal effective concentration” (EC 50 ) value, which refers to the concentration of an antibody where 50%of its maximal effect (e.g., binding or inhibition etc. ) is observed.
- the EC 50 value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assays.
- the anti-CD4 anti-PD-L1 bispecific antibody provided herein comprises a CD4 binding domain and a PD-L1 binding domain.
- Each of the CD4 binding domain and PD-L1 binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-CD4 or anti-PD-L1 antibody disclosed herein.
- CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or unchangeable. In other words, it is possible to replace or change or modify one or more CDRs disclosed herein, yet substantially retain the specific binding affinity to CD4 or PD-L1.
- the CD4 binding domain and PD-L1 binding domain have a CDR sequence as listed in Table 1 below.
- the CD4 binding domain and PD-L1 binding domain provided herein comprise suitable framework region (FR) sequences, as long as the domain can specifically bind to CD4 or PD-L1.
- FR framework region
- the CDR sequences provided in Table 1 can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.
- the CD4 binding domain and PD-L1 binding domain provided herein are humanized.
- a humanized binding domain is desirable in its reduced immunogenicity in human.
- a humanized domain is chimeric, as non-human CDR sequences are grafted to human or substantially human FR sequences.
- Humanization of a binding domain can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al., Nature (1986) 321: 522-525; Riechmann et al., Nature (1988) 332: 323-327; Verhoeyen et al., Science (1988) 239: 1534-1536) .
- Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art.
- “best-fit” approach can be used, where a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as the human scaffold for grafting the non-human CDR sequences (see, for example, Sims et al., J. Immunol. (1993) 151: 2296; Chothia et al., J. Mot. Biol. (1987) 196: 901) .
- a framework derived from the consensus sequence of all human antibodies may be used for the grafting of the non-human CDRs (see, for example, Carter et at. Proc. Natl. Acad. Sci. USA (1992) 89:4285; Presta et al., J. Immunol. (1993) 151: 2623) .
- the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences except for the CDR sequences which are non-human.
- the variable region FRs, and constant regions if present are entirely or substantially from human immunoglobulin sequences.
- the human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody.
- the CD4 binding domain and PD-L1 binding domain provided herein comprise paired heavy chain and light chain variable region amino acid sequences as provided in Table 2 below.
- the CD4 binding domain or PD-L1 binding domain is a single chain variable fragment (svFv) .
- a scFv is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker.
- the linker comprises an amino acid sequence of SEQ ID NO. 19. This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered.
- scFv can be created directly from subcloned heavy and light chains derived from a hybridoma.
- Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.
- Flexible linkers generally are comprised of helix-and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well.
- Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries.
- a random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition.
- the scFv repertoire (approx. 5 ⁇ 10 6 different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity.
- bispecific antibodies and antigen-binding fragments thereof provided herein can be in a suitable format known in the art.
- an exemplary bispecific format can be IgG-scFv fusions, dual variable domain (DVD) -Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.
- bispecific molecules can be in symmetric or asymmetric architecture.
- the bispecific antibodies and the fragments thereof provided herein further comprise an immunoglobulin constant region.
- an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region.
- the heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions.
- the heavy chain constant region comprises an Fc region.
- the light chain constant region comprises C ⁇ or C ⁇ .
- the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region.
- the heavy chain constant region comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the heavy chain constant region comprises a constant region of human IgG1.
- the PD-L1 binding domain is a scFv (single chain fragment variable) domain.
- the scFv domain is linked to a heavy chain constant region.
- the scFv domain is linked to a light chain constant region.
- the anti-CD4 anti-PD-L1 bispecific antibody has a configuration illustrated in FIG. 1. As shown in FIG. 1, the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region.
- the PD-L1 binding domain is a scFv domain linked to a heavy chain constant region.
- the anti-CD4 anti-PD-L1 bispecific antibody has a configuration illustrated in FIG. 2. As shown in FIG. 2, the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region.
- the PD-L1 binding domain is a scFv domain linked to a light chain constant region.
- the anti-CD4 anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 21; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 10.
- the anti-CD4 and anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 9; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 22.
- bispecific antibodies and antigen-binding fragments provided herein can be made with any suitable methods known in the art.
- two immunoglobulin heavy chain-light chain pairs having different antigenic specificities are co-expressed in a host cell to produce bispecific antibodies in a recombinant way (see, for example, Milstein and Cuello, Nature, 305: 537 (1983) ) , followed by purification by affinity chromatography.
- the antibodies and antigen-binding fragments thereof provided herein also encompass various variants thereof.
- the antibodies and antigen-binding fragments thereof encompasses various types of variants of an exemplary antibody provided herein.
- the antibody variants comprise one or more modifications or substitutions in one or more CDR sequences as provided in Table 1, one or more variable region sequences (but not in any of the CDR sequences) provided herein, and/or the constant region (e.g., Fc region) .
- Such variants retain specific binding affinity to CD4 or PD-L1 of their parent antibodies, but have one or more desirable properties conferred by the modification (s) or substitution (s) .
- the antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamidation or deamination, improved or increased effector function (s) , reduced or depleted effector function (s) , improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g., one or more introduced cysteine residues) .
- the parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085) .
- target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
- a neutral or negatively charged amino acid e.g., alanine or polyalanine
- substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution.
- the potential residues may be further assessed by substituting with a different type of residue (e.g. cysteine residue, positively charged residue, etc. ) .
- Affinity variant may contain modifications or substitutions in one or more CDR sequences, one or more FR sequences, or the heavy or light chain variable region sequences provided herein.
- the affinity variants retain specific binding affinity to CD4 or PD-L1 of the parent antibody, or even have improved CD4 or PD-L1 specific binding affinity over the parent antibody.
- a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to human CD4 or PD-L1.
- computer software can be used to virtually simulate the binding of the antibodies to human CD4 or PD-L1 and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity or targeted for substitution to provide for a stronger binding.
- the humanized antibody or antigen-binding fragment provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences.
- an affinity variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the CDR sequences and/or FR sequences in total.
- the bispecific antibodies and antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1, and in the meantime retain the binding affinity to CD4 and PD-L1 at a level similar to or even higher than its parent antibody.
- the bispecific antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) provided herein, and in the meantime retain the binding affinity to CD4 and PD-L1 at a level similar to or even higher than its parent antibody.
- the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) .
- the antibody comprises a particular glycosylation pattern.
- an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation) .
- the glycosylation pattern of an antibody may be altered to, for example, increase the affinity or avidity of the antibody for an antigen.
- modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody sequence.
- one or more amino acid substitutions can be made that result removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
- Such aglycosylation may increase the affinity or avidity of the antibody for antigen. See, e.g., U.S. Patents 5,714,350 and 6,350,861.
- an antibody may also be made in which the glycosylation pattern includes hypofucosylated or afucosylated glycans, such as a hypofucosylated antibodies or afucosylated antibodies have reduced amounts of fucosyl residues on the glycan.
- the antibodies may also include glycans having an increased amount of bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such modifications can be accomplished by, for example, expressing the antibodies in a host cell in which the glycosylation pathway was been genetically engineered to produce glycoproteins with particular glycosylation patterns.
- the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 ( ⁇ (1, 6) -fucosyltransferase) , such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates.
- the Ms704, Ms705, and Ms709 FUT8-/-cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704.
- EP 1 176 195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the ⁇ -1, 6 bond-related enzyme.
- EP 1 176 195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662) .
- PCT Publication WO 2003/035835 describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell.
- Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231.
- antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna (US Patent 7, 632, 983) . Methods for production of antibodies in a plant system are disclosed in the U.S. Patents 6,998,267 and 7,388,081.
- PCT Publication WO1999/054342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., ⁇ (1, 4) -N-acetylglucosaminyltransferase III (GnTIII) ) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies. Hypofucosylation is also called afucosylation when fucosylation is minimal on antibodies.
- glycoprotein-modifying glycosyl transferases e.g., ⁇ (1, 4) -N-acetylglucosaminyltransferase III (GnTIII)
- GnTIII glycoprotein-modifying glycosyl transferases
- Hypofucosylation is also called afucosylation when fucosylation is minimal on antibodies.
- the fucose residues of the antibodies can be cleaved off using a fucosidase enzyme; e.g., the fucosidase ⁇ -L-fucosidase removes fucosyl residues from antibodies.
- a fucosidase enzyme e.g., the fucosidase ⁇ -L-fucosidase removes fucosyl residues from antibodies.
- Antibodies disclosed herein further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian-or human-like glycosylation patterns.
- a particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Patents 7,029,872 and 7,449,308) .
- These genetically modified host cells have been used to produce antibodies that have predominantly particular N-glycan structures.
- fungi such as yeast or filamentous fungi lack the ability to produce fucosylated glycoproteins
- antibodies produced in such cells will lack fucose unless the cells are further modified to include the enzymatic pathway for producing fucosylated glycoproteins (See for example, PCT Publication WO2008112092) .
- the antibodies disclosed herein further include those produced in lower eukaryotic host cells and which comprise fucosylated and nonfucosylated hybrid and complex N-glycans, including bisected and multiantennary species, including but not limited to N-glycans such as GlcNAc (1-4)Man3GlcNAc2; Gal (1-4) GlcNAc (1-4) Man3GlcNAc2; NANA (1-4) Gal (1-4) GlcNAc (1-4)Man3GlcNAc2.
- N-glycans such as GlcNAc (1-4)Man3GlcNAc2; Gal (1-4) GlcNAc (1-4) Man3GlcNAc2; NANA (1-4) Gal (1-4) GlcNAc (1-4)Man3GlcNAc2.
- the antibody compositions provided herein may comprise antibodies having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2.
- the hybrid N-glycan is the predominant N-glycan species in the composition.
- the hybrid N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%of the hybrid N-glycans in the composition.
- the antibody compositions provided herein comprise antibodies having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2.
- the complex N-glycan is the predominant N-glycan species in the composition.
- the complex N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%of the complex N-glycans in the composition.
- the N-glycan is fusosylated.
- the fucose is in an ⁇ 1, 3-linkage with the GlcNAc at the reducing end of the N-glycan, an ⁇ 1, 6-linkage with the GlcNAc at the reducing end of the N-glycan, an ⁇ 1, 2-linkage with the Gal at the non-reducing end of the N-glycan, an ⁇ 1, 3-linkage with the GlcNac at the non-reducing end of the N-glycan, or an ⁇ 1, 4-linkage with a GlcNAc at the non-reducing end of the N-glycan.
- the glycoform is in an ⁇ 1, 3-linkage or ⁇ 1, 6-linkage fucose to produce a glycoform selected from the group consisting of Man5GlcNAc2 (Fuc) , GlcNAcMan5GlcNAc2 (Fuc) , Man3GlcNAc2 (Fuc) , GlcNAcMan3GlcNAc2 (Fuc) , GlcNAc2Man3GlcNAc2 (Fuc) , GalGlcNAc2Man3GlcNAc2 (Fuc) , Gal2GlcNAc2Man3GlcNAc2 (Fuc) , NANAGal2GlcNAc2Man3GlcNAc2 (Fuc) , and NANA2Gal2GlcNAc2Man3GlcNAc2 (Fuc) ; in an ⁇ 1, 3-linkage or ⁇ 1, 4-linkage
- the antibodies comprise high mannose N-glycans, including but not limited to, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or N-glycans that consist of the Man3GlcNAc2 N-glycan structure.
- the complex N-glycans further include fucosylated and non-fucosylated (or afucosylated) bisected and multiantennary species.
- N-glycan and “glycoform” are used interchangeably and refer to an N-linked oligosaccharide, for example, one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide.
- N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
- bispecific antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the antibody or antigen binding fragment.
- the antibody or antigen binding fragment thereof may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached.
- Glycosylation of antibodies is typically either N-linked or O-linked.
- N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline.
- O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
- the anti-CD4 and anti-PD-L1 bispecific antibodies and antigen-binding fragments provided herein also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
- a free cysteine residue is one which is not part of a disulfide bridge.
- a cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl.
- Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
- the antibodies disclosed herein can also be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity) .
- the antibodies disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
- the numbering of residues in the Fc region is that of the EU index of Kabat.
- the antibodies disclosed herein also include antibodies with modified (or blocked) Fc regions to provide altered effector functions.
- Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions) , glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region.
- the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is increased or decreased.
- the number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
- the antibody is modified to increase its biological half-life.
- one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent 6,277,375.
- the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patents 5,869,046 and 6,121,022.
- the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function (s) of the antibodies.
- one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen binding ability of the parent antibody.
- the effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patents 5,624,821 and 5,648,260.
- one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO1994/029351.
- the Fc region is modified to increase or decrease the ability of the antibodies to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase or decrease the affinity of the antibodies for an Fc ⁇ receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340,
- the Fc region is modified to decrease the ability of the antibodies to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.
- the Fc region is modified to abolish the ability of the antibodies to mediate effector function by modifying residues 234, 235 and 329 to alanine or glycine (L234A-L235A-P329G) .
- the bispecific antibodies and antigen-binding fragments provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region.
- the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) that improves pH-dependent binding to neonatal Fc receptor (FcRn) .
- FcRn neonatal Fc receptor
- Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell.
- Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D.
- the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) that alters the antibody-dependent cellular cytotoxicity (ADCC) .
- Certain amino acid residues at CH2 domain of the Fc region can be substituted to provide for enhanced ADCC activity.
- carbohydrate structures on the antibody can be changed to enhance ADCC activity.
- the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) that alters Complement Dependent Cytotoxicity (CDC) , for example, by improving or diminishing C1q binding and/or CDC (see, for example, WO99/51642; Duncan &Winter Nature 322: 738-40 (1988) ; U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821) ; and WO1994/029351 concerning other examples of Fe region variants.
- CDC Complement Dependent Cytotoxicity
- the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) in the interface of the Fc region to facilitate and/or promote heterodimerization.
- modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex.
- Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731,168.
- anti-CD4 anti-PD-L1 bispecific antigen-binding fragments are also provided herein.
- Various types of antigen-binding fragments are known in the art and can be developed based on the bispecific antibodies provided herein, including for example, the exemplary antibodies whose CDR and variable sequences are provided herein, and their different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants and so on) .
- an anti-CD4 anti-PD-L1 bispecific antigen-binding fragment is a camelized single domain antibody, a diabody, a single chain Fv fragment (scFv) , an scFv dimer, a BsFv, a dsFv, a (dsFv) 2 , a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2 , a bispecific antibody, a ds diabody, a nanobody, a domain antibody, a single domain antibody, or a bivalent domain antibody.
- the bispecific antibodies and antigen-binding fragments thereof further comprise a conjugate moiety.
- the conjugate moiety can be linked to the antibodies and antigen-binding fragments thereof.
- a conjugate moiety is a proteinaceous or non-proteinaceous moiety that can be attached to the antibody or antigen-binding fragment thereof. It is contemplated that a variety of conjugate moieties may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugate moieties may be linked to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
- the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugate moieties.
- a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate moiety.
- the antibodies may be linked to a conjugate moiety indirectly, or through another conjugate moiety.
- the antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin.
- conjugate moiety examples include without a limitation an immune-modulatory agent, an anti-tumor drug, a STING (Stimulator of Interferon Genes) agonist, a cytokine, a clearance-modifying agent, a toxin (e.g., a chemotherapeutic agent) , an immune cell stimulator (e.g., a TLR agonist) , a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label) , a DNA, an RNA, or purification moiety.
- an immune-modulatory agent an anti-tumor drug
- STING Stimulator of Interferon Genes
- cytokine e.g., a chemotherapeutic agent
- a clearance-modifying agent e.g., a chemotherapeutic agent
- an immune cell stimulator e.g., a TLR
- immune modulatory agent examples include without limitation an immune modulator molecule disclosed herein (e.g., PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, Fc receptors, FCRL (1-6) , A2AR, CD160, 2B4, TGF- ⁇ , TGF- ⁇ R, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA (1-6) , OX40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-1, clever-1/stabilin-1, ADGRE, TREM1, TREM2, CD122, ICAM-1, IDO, NKG2D/C, SLAMF7, MS4A4A, SIGLEC (7-15) , NKp80, NKG2A, CD160, CD161, CD300, CD163, B7-H3, LFA-1, ICOS, 4-1BB, GITR, BAFFR, HVEM,
- the immune modulatory agent linked to the antibodies and antigen-binding fragments disclosed herein is a ligand-binding protein (e.g., ligand trapper) specific to an immune modulatory receptor.
- a ligand-binding protein e.g., ligand trapper
- anti-tumor drugs include without limitation a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenesis agent, a cancer immunotherapeutic agent, a apoptotic agent, an anti-tubulin agent, an anti-HER-2 antibody, an anti-CD20 antibody, an epidermal growth factor receptor (EGFR) antagonist, HER1/EGFR inhibitor, a platelet derived growth factor inhibitor, a COX-2 inhibitor, an interferon, a CTLA4 inhibitor (e.g., anti-CTLA antibody ipilimumab or tremelimumab) , a PD-l or PD-L1 inhibitor (e.g., or nivolumab, or pembrolizumab, or atezolizumab, or avelumab, or durvalumab, or cemiplimab-rwlc, or sintilimab, tislelizumab
- a “toxin” can be any agent that is detrimental to cells or that can damage or kill cells.
- toxin include, without limitation, taxol, cytochalasin B, deruxtecan, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF) , mertansine, emtansine, DM1, maytansinoid DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate,
- detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or ⁇ -D-galactosidase) , radioisotopes (e.g.
- the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody.
- Illustrative examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like.
- the polymer may be of any molecular weight, and may be branched or unbranched.
- the number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules.
- the conjugate moiety can be a purification moiety such as a magnetic bead.
- the antibodies and antigen-binding fragments thereof provided herein is used for a base for a conjugate.
- the present disclosure provides isolated polynucleotides that encode the bispecific antibodies and antigen-binding fragments thereof disclosed herein.
- the isolated polynucleotides comprise one or more nucleotide sequences listed in Table 1 that encodes the variable region of the exemplary antibodies provided herein.
- DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) .
- the encoding DNA may also be obtained by synthetic methods.
- the isolated polynucleotide that encodes the bispecific antibodies and antigen-binding fragments can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
- Many vectors are available.
- the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1 ⁇ ) , and a transcription termination sequence.
- the present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibodies or antigen-binding fragments, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
- promoter e.g., SV40, CMV, EF-1 ⁇
- vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBA
- RTM. pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
- Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression.
- Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
- Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E.
- eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for bispecific antibody-encoding vectors.
- Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
- a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424) , K. bulgaricus (ATCC 16,045) , K. wickeramii (ATCC 24,178) , K.
- waltii ATCC 56,500
- K. drosophilarum ATCC 36,906
- K. thermotolerans K. marxianus
- yarrowia EP 402,226)
- Pichia pastoris EP 183,070
- Candida Trichoderma reesia
- Neurospora crassa Neurospora crassa
- Schwanniomyces such as Schwanniomyces occidentalis
- filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
- Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms.
- invertebrate cells include plant and insect cells.
- Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruit fly) , and Bombyx mori have been identified.
- a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
- Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
- the host cell is 293
- Host cells are transformed with the above-described expression or cloning vectors for bispecific antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
- the antibody may be produced by homologous recombination known in the art.
- the host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media.
- Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells.
- any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
- the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology (1992) 10: 163-167 describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
- sodium acetate pH 3.5
- EDTA EDTA
- PMSF phenylmethylsulfonylfluoride
- Cell debris can be removed by centrifugation.
- supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
- a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
- the bispecific antibodies and antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.
- Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof.
- the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
- Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. (1983) 62: 1-13) .
- Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. (1986) 5: 1567-75) .
- the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
- Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
- the antibody comprises a CH3 domain
- the Bakerbond ABX TM resin J. T. Baker, Phillipsburg, N.J. ) is useful for purification.
- the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .
- the antibodies of the present disclosure may be purified.
- purified as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state.
- a purified protein therefore also refers to a protein, free from the environment in which it may naturally occur.
- substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%or more of the proteins in the composition.
- Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) . Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing.
- protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.
- polypeptide In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions.
- the polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide.
- affinity column which binds to a tagged portion of the polypeptide.
- antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody.
- agents i.e., protein A
- antigens may be used to simultaneously purify and select appropriate antibodies.
- Such methods often utilize the selection agent bound to a support, such as a column, filter or bead.
- the antibodies are bound to a support, contaminants removed (e.g., washed away) , and the antibodies released by applying conditions (salt, heat, etc. ) .
- compositions comprising the bispecific antibodies or antigen-binding fragments thereof described herein and one or more pharmaceutically acceptable carriers.
- Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
- Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins.
- Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate.
- compositions that comprise one or more antibodies or antigen-binding fragments as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
- pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (
- Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
- Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
- Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
- compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.
- Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
- the pharmaceutical compositions are formulated into an injectable composition.
- the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
- Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
- the solutions may be either aqueous or nonaqueous.
- unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
- a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent.
- the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents.
- the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
- the resulting solution will be apportioned into vials for lyophilization.
- Each vial can contain a single dosage or multiple dosages of the bispecific antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
- the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
- Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
- the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
- the pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof described herein further comprise one or more additional therapeutic agents that are co-administered with the bispecific antibodies or antigen-binding fragments thereof.
- additional therapeutic agents are disclosed infra in Section IV. It can be understood that the additional therapeutic agents can be co-formulated with the bispecific antibodies or antigen-binding fragments thereof, or be mixed with the bispecific antibodies or antigen-binding fragments thereof right before the administration, such as in the IV infusion bag.
- the present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing cancer.
- Solid tumors include but are not limited to, non-small cell lung cancer (squamous/non-squamous) , small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma) , pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, multiple myeloma, mycoses fungoides, Merkel cell cancer, hepatocellular carcinoma (HCC) , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synoviom
- HCC hepatocellular carcinoma
- Solid tumors are characterized by multiple biologic hallmarks including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, tumor promoting inflammation, avoiding immune destruction, genomic instability and mutation, and deregulating cellular energetics.
- Treatment efforts have evolved from cytotoxic chemotherapies targeting rapidly dividing cells to small molecules inhibiting select signaling pathways to monoclonal antibodies targeting surface proteins. More recently the concept of cancer immunotherapy to reinvigorate endogenous immunity or cellular therapies utilizing synthetic immunity have shown promise.
- immune checkpoint inhibitors such as anti-CTLA-4 or anti-PD-1/PD-L1 have led to long-term progression-free and overall survival in a minority of patients.
- Hematologic malignancies include but are not limited to acute lymphocytic/lymphoblastic leukemia (ALL) , acute myeloid leukemia (AML) , B-cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN) , chronic lymphoblastic leukemia (CLL) , chronic lymphocytic leukemia (CLL) , chronic myeloid leukemia (CML) , chronic myelomonocytic leukemia (CMML) , classical Hodgkin lymphoma (CHL) , diffuse large B-cell lymphoma (DLBCL) , extranodal NK/T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8-associated primary effusion lymphoma, lymphoid malignancy, multiple myeloma (MM) , myelodysplasia, myelodysplastic syndrome (MDS) , non-Hod
- an antibody or antigen-binding fragment as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
- the antibody or antigen-binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.0001 mg/kg to about 100 mg/kg. In certain of these embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
- Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) .
- a single dose may be administered, or several divided doses may be administered over time.
- the antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
- parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
- non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal, or topical routes.
- the antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents.
- the antibodies or antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, for example, a chemotherapeutic agent or an anti-cancer drug.
- an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen-binding fragment and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition.
- an antibody or antigen-binding fragment administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent.
- An antibody or antigen-binding fragment administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and second agent are administered via different routes.
- additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Prescriber’s Digital Reference (available online only at pdr. net) or protocols well known in the art.
- the agent for combination therapy is an anti-neoplastic composition.
- an “anti-neoplastic composition” refers to a composition useful in treating cancer comprising at least one active therapeutic agent.
- therapeutic agents include, but are not limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, cancer immunotherapeutic agents, apoptotic agents, anti-tubulin agents, and other-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor) , HER1/EGFR inhibitor (e.g., erlotinib platelet derived growth factor inhibitors (e.g., (Imatinib Mesylate) ) , a COX-2 inhibitor (e.g., celecoxib) , interferons, CTLA4 inhibitors
- EGFR epiderma
- the agent for combination therapy is a chemotherapeutic agent.
- a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer.
- examples of chemotherapeutic agents that can be administered in methods herein include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone) ; a camptothecin (including the synthetic analogue topotecan) ; bryostatin; call
- dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores) , aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) , epirubicin,
- chemotherapeutic agents that can be administered in methods herein include anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens and selective estrogen receptor modulators (SERMs) , including, for example, tamoxifen (including tamoxifen) , raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, le
- the agent for combination therapy is an anti-angiogenesis agent.
- an “anti-angiogenesis agent” refers to a small molecular weight substance, a polynucleotide (including, e.g., an inhibitory RNA (RNAi or siRNA) ) , a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly.
- RNAi or siRNA inhibitory RNA
- the anti-angiogenesis agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor.
- an anti-angiogenesis agent that can be administered in methods herein can include an antibody or other antagonist to an angiogenic agent, e.g., antibodies to VEGF-A (e.g., bevacizumab ) or to the VEGF-A receptor (e.g., KDR receptor or Flt-l receptor) , anti-PDGFR inhibitors such as (Imatinib Mesylate) , small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, /SUl 1248 (sunitinib malate) , AMG706, or those described in, e.g., international patent application WO 2004/113304) .
- an antibody or other antagonist to an angiogenic agent e.g., antibodies to VEGF-A (e.g., bevacizumab ) or to the VEGF-A receptor (e.g., KDR receptor or Flt-l receptor)
- anti-PDGFR inhibitors
- Anti-angiogenesis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’A more (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22: 3172-3179; Ferrara &Alitalo (1999) Nature Medicine 5 (12) : 1359-1364; Tonini et al. (2003) Oncogene 22: 6549-6556; and Sato (2003) Int. J.Clin. Oncol. 8: 200-206.
- native angiogenesis inhibitors e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’A more (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22: 3172-3179; Ferrara &Alitalo (1999) Nature Medicine
- the agent for combination therapy is a growth inhibitory agent.
- a “growth inhibitory agent” as used herein refers to a compound or composition that inhibits growth of a cell (such as a cell expressing VEGF) either in vitro or in vivo.
- the growth inhibitory agent that can be administered in methods herein may be one that significantly reduces the percentage of cells (such as a cell expressing VEGF) in S phase.
- growth inhibitory agents include, but are not limited to, agents that block cell cycle progression (at a place other than S phase) , such as agents that induce Gl arrest and M-phase arrest.
- Classical M-phase blockers include the vincas (vincristine and vinblastine) , taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
- Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
- Taxanes are anticancer drugs both derived from the yew tree.
- Docetaxel Rhone-Poulenc Rorer
- paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
- the dose of the agent for the combination therapy can be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular agent. Typically, the attending physician will decide the dosage of the agent for the combination therapy with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, the agent be administered, route of administration, and the severity of the condition being treated.
- the dose for the combination therapy can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg bodyweight/day. Dosage units may be also expressed in mg/m2, which refer to the quantity in milligrams per square meter of body surface area.
- Each therapeutic agent in the combination therapy described herein may be administered simultaneously (e.g., in the same medicament or at the same time) , concurrently (i.e., in separate medicaments administered one right after the other in any order or sequentially in any order.
- Sequential administration may be useful when the therapeutic agents in the combination therapy are in different dosage forms (one agent is a tablet or capsule and another agent is a sterile liquid) and/or are administered on different dosing schedules, e.g., a chemotherapeutic that is administered at least daily and a biotherapeutic that is administered less frequently, such as once weekly, once every two weeks, or once every three weeks.
- the bispecific antibody of the present disclosure and the second drug are combined or co-formulated in a single dosage form. In certain embodiments, the bispecific antibody of the present disclosure and the second drug are administered separately. Although the simultaneous administration of the bispecific antibody of the present disclosure and the second drug may be maintained throughout a period of treatment, anti-cancer activity may also be achieved by subsequent administration of one compound in isolation (for example, the bispecific antibody following initial combination treatment, or alternatively, the second drug following initial combination treatment) . In some embodiments, the bispecific antibody is administered before administration of the second drug, while in other embodiments, the bispecific antibody is administered after administration of the second drug.
- At least one of the therapeutic agents in the combination therapy is administered using the same dosage regimen (dose, frequency and duration of treatment) that is typically employed when the agent is used as monotherapy for treating the same cancer.
- the patient receives a lower total amount of at least one of the therapeutic agents in the combination therapy than when the agent is used as monotherapy, e.g., smaller doses, less frequent doses, and/or shorter treatment duration.
- the combination therapy of the invention may be used prior to or following surgery to remove a tumor and may be used prior to, during or after radiation therapy.
- the combination therapy of the invention may be used to treat a tumor that is large enough to be found by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan.
- the combination therapy of the invention is used to treat an advanced stage tumor having dimensions of at least about 200 mm 3 , 300 mm 3 , 400 mm 3 , 500 mm 3 , 750 mm 3 , or up to 1000 mm 3 .
- the present disclosure also provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating cancer in a subject.
- Example 1 Construction and expression of anti-CD4 and anti-PD-L1 bispecific antibodies
- anti-CD4 antibody is from patent US Patent No. 11,254,745 B1, disclosure of which is incorporated herein by reference in their entirety.
- sequence of anti-PD-L1 antibody is from patent US Patent No. 8,217,149 B2, disclosure of which is incorporated herein by reference in their entirety.
- Anti-CD4 and anti-PD-L1 bispecific antibodies were constructed by fusing the single chain variable fragment (scFv) of anti-PD-L1 antibody (atezolizumab) to the carboxyl-terminus of heavy chain (2B6-hIgG1/Atezo-scFv) (FIG. 1) or light chain (2B6-hkappa/Atezo-scFv) (FIG.
- Freestyle 293 cells 1000 mL at 10 6 /mL were transfected with 1000 ⁇ g of each of the heavy and light chain expression plasmids and cultured for 6 days at 37°C.
- the bispecific antibody in the supernatant was then purified with Protein-A column (GE healthcare) .
- Example 2 ELISA based binding analysis of anti-CD4/anti-PD-L1 bispecific antibody
- ELISA binding analysis was conducted by using human CD4-mFc or PD-L1-mFc as antigen.
- 96-well plates (Costar, Cat No: 9018) were coated with 100 ⁇ L of 2 ⁇ g/ml CD4-mFc or PD-L1-mFc in PBS coating buffer (Hyclone, Cat No: SH30256.01B) overnight at 4°C.
- the wells were aspirated and non-specific binding sites were blocked by adding 200 ⁇ L of blocking buffer (PBS with 1% (w/v) of bovine serum albumin (BSA, Roche, Cat No: 738328) ) and incubating for 1 hour at 37°C.
- BSA bovine serum albumin
- 2B6-hIgG1/Atezo-scFv and 2B6-hkappa/Atezo-scFv bispecific antibody can bind to both CD4 and PD-L1 with an EC50 comparable to the corresponding monoclonal antibody.
- Example 3 Binding kinetic study of anti-CD4/anti-PD-L1 bispecific antibody with CD4 and PD-L1
- the binding kinetics of bispecific antibody with CD4 and PD-L1 were measured by surface plasmon resonance (SPR) analysis, which was performed at 25°C on a Biacore T200 instrument.
- Protein A GE, Cat. No: 29139131-AA
- GE, BR10050 an amine coupling kit
- diluted bispecific antibody 1.5 ⁇ g/mL was injected over experiment flow cell for 1 minute to be captured.
- CD4-HisTag and PD-L1-HisTag analyte series were prepared by diluting the stocks with running buffer to 100 nM followed by 2-fold serial dilution in the same buffer down to 0.78 nM.
- Analytes were injected in series over the reference and experiment flow cells for 3 minutes at a flow rate of 30 ⁇ L/minute.
- Running buffer PBS with 0.05%P20
- the biosensor surface was regenerated with 3-minute injection of 10 mM pH 2.0 Glycine-HCl buffer at a flow rate of 10 ⁇ L/minute.
- For each analyte sample injection i.e.
- binding responses obtained from the experimental biosensor surface were double referenced by subtracting simultaneously recorded responses from the reference surface followed by additional subtraction of responses from a single referenced running buffer sample.
- the association and dissociation rate constants (ka and kd) were determined simultaneously by fitting double-referenced sensorgrams of the entire titration series to Langmuir model (1: 1) using Biaevaluation software.
- the binding affinity of anti-CD4/anti-PD-L1 bispecific antibody with human CD4-HisTag and PD-L1-HisTag are summarized in Table 5-6.
- Example 4 Effect of anti-CD4/anti-PD-L1 bispecific antibody on T cell activation in the mixed lymphocyte reaction
- Human CD4 + T-cells were purified from human PBMC using a CD4 + negative selection isolation kit (Mitenyi Biotech, Cat No: 130-091-155) .
- Immature dendritic cells (DC) were derived from monocytes isolated from human PBMC using the Mo-DC Generation Toolbox (Miltenyi, Cat No: 130-093-568) .
- the cells were cultured with Mo-DC Differentiation Medium for 7 days, and were then induced to be mature DC with Mo-Dc Maturation medium for 2 days. For mixed lymphocyte reaction setting-up, each reaction was added with 10 5 purified T-cells and 10 4 allogeneic mature DC cells in a total volume of 200 ⁇ L.
- the bispecific antibodies were assayed at different concentrations (0.02, 0.2, 2, 20 ⁇ g/mL) , and an IgG1 isotype control antibody was used as a negative control.
- the cells were cultured for 5 days at 37 °C. On day 3, the levels of IFN- ⁇ and IL-2 in the culture medium were measured using the IL-2 ELISA kit (eBioscience) and hIFN- ⁇ ELISA kit (R&D, Cat No: DY285) . The results are shown in FIG. 5 for IL-2 secretion, and FIG. 6 for IFN- ⁇ secretion.
- Example 5 Antibody-dependent cell-mediated cytotoxicity effect of anti-CD4/anti-PD-L1 bispecific antibody
- anti-CD4/anti-PD-L1 bispecific antibody was assessed based on the killing of CD4 positive cells in PBMC.
- PBMCs (2 ⁇ 10 ⁇ 5 cells/well) were incubated with series concentrations of anti-CD4/anti-PD-L1 bispecific 2 antibody (160 ng/mL, 16 ng/mL, 5.333 ng/mL, 1.778ng/mL, 0.178 ng/mL, 0.018 ng/mL) at 37°C, 5%CO2 for 24 hours.
- AF-488 labeled CD4 antibody (OKT4-AF488, the binding epitope of OKT4 antibody does not overlap with the CD4 antibody 2B6) was then added to each well and incubated at 4 °C for 1 hour, and living CD4+ cells in each well were detected with FACS.
- the ADCC effect was shown in FIG. 7 by calculating with the following formula:
- ADCC% (1-No. of living CD4 positive cells in the presence of antibody/No. of living CD4 positive cells in the absence of antibody) ⁇ 100%
- 2B6-hIgG1/Atezo-scFv and 2B6-hkappa/Atezo-scFv bispecific antibody can induce ADCC with an EC50 comparable to the corresponding monoclonal antibody 2B6.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Abstract
Provided are anti-CD4 and anti-PD-L1 bispecific antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding the same, pharmaceutical compositions comprising the same, and the uses thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
The present application claims the priority of PCT Application No. PCT/CN2022/074181, filed January 27, 2022, the disclosure of which is incorporated herein by reference in the entirety.
SEQUENCE LISTING
The sequence listing that is contained in the file named “056211-8005WO02_seq list. xml” , which is 30,433 bytes (as measured in Microsoft Windows) and was created on January 25, 2023, is filed herewith by electronic submission and is incorporated by reference herein.
The present disclosure relates generally to the fields of medicine, oncology, and immunology. More particular, the disclosure relates to bispecific antibodies that bind to CD4 and PD-L1 and to methods of using such bispecific antibodies.
CD4 is a monomeric type I transmembrane glycoprotein comprising four extracellular Ig-like domains, a hydrophobic transmembrane domain, and a highly basic cytoplasmic tail (Glatzova D, et al., Front Immunol (2019) 10: 618) . The N-terminal Ig-like domains of CD4 interact with the α2 and β2 domains of MHC class II molecules to assemble TCR–pMHC–CD4 ternary complex, and the resulting close proximity between the TCR and CD4 allows the tyrosine kinase Lck bound to the cytoplasmic tail of CD4 to phosphorylate tyrosine residues of immunoreceptor tyrosine activation motifs (ITAMs) on the cytoplasmic domains of CD3 to amplify the signal generated by the TCR (Rudd CE, et al., Proc Natl Acad Sci USA (1988) 85: 5190; Barber EK, et al., Proc Natl Acad Sci USA (1989) 86: 3277 and Li QJ, et al., Nat Immunol (2004) 5: 791) .
CD4 is expressed in a large proportion of thymocytes (80–90%) and over 50%of the peripheral blood T-cells (Nakamura K, et al., Mol Immunol (2003) 39: 909) . CD4 is also expressed to a minor extent on macrophages, monocytes, dendritic cells and Langerhans cells. CD4 plays an important role not only in the differentiation of thymocytes and the regulation of T-lymphocyte/B-lymphocyte adhesion, but also in MHC class II-restricted T cell activation
(Gaubin M, et al., Eur J Clin Chem Clin Biochem (1996) 34: 723; Artyomov MN, et al., Proc Natl Acad Sci USA (2010) 107: 16916; Yili L, et al., Front Immunol (2013) 4: 206) . More importantly, CD4 expressing cells, such as regulatory T cells (Tregs) have been shown to suppress tumor-induced immunity, thereby compromising the therapeutic efficacy of various cancer therapies (Baba J, et al., Blood (2012) 120: 2417; Togashi Y, et al., Nature Reviews Clinical Oncology (2019) 16: 356) .
Programmed death-ligand 1 (PD-L1) is a type 1 transmembrane protein that plays a major role in suppressing the adaptive immune response. Normally the adaptive immune system reacts to antigens that are associated with immune system activation by exogenous or endogenous danger signals. In turn, clonal expansion of antigen-specific CD8+ T cells and/or CD4+ helper cells is propagated. The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM) . This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells.
Recent studies have suggested that high level expression of PD-L1 in tumor cells and high density of CD4+ Treg cells may indicate a tumor microenvironment that promote tumor cell survival (Zhang et al. BMC Cancer (2020) 20: 127) . Therefore, there is a need for developing new compositions of tumor therapeutics that can target both CD4 and PD-L1.
BRIEF SUMMARY OF THE INVENTION
The present disclosure provides anti-CD4 and anti-PD-L1 bispecific antibodies and antigen-binding fragment thereof, amino acid and nucleotide sequences thereof, and uses thereof.
In one aspect, the present disclosure provides an anti-CD4 and anti-PD-L1 bispecific antibody or an antigen-binding fragment thereof. In some embodiments, the anti-CD4 and anti-PD-L1 bispecific antibody or an antigen-binding fragment comprises a CD4 binding domain and a PD-L1 binding domain. In some embodiments, the CD4 binding domain comprise (i) a heavy chain (HC) variable region (VH) comprising a HCDR1 comprising the sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the sequence set forth in SEQ ID NO:2, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 3, and variants thereof wherein one or more of the HC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof, and (ii) a light chain (LC) variable region (VL) comprising
a LCDR1 comprising the sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the sequence set forth in SEQ ID NO: 6, and variants thereof wherein one or more of the LC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof. In some embodiments, the PD-L1 binding domain comprises (i) a VH comprising a HCDR1 comprising the sequence set forth in SEQ ID NO: 11, a HCDR2 comprising the sequence set forth in SEQ ID NO: 12, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 13, and variants thereof wherein one or more of the HC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof, and (ii) a VL comprising a LCDR1 comprising the sequence set forth in SEQ ID NO: 14, a LCDR2 comprising the sequence set forth in SEQ ID NO: 15, and a LCDR3 comprising the sequence set forth in SEQ ID NO: 16, and variants thereof wherein one or more of the LC-CDRs has one, two, or three amino acid substitutions, additions, deletions or a combination thereof. In some embodiments, the VH of the CD4 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 7, and the VL of the CD4 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 8. In some embodiments, the VH of the PD-L1 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 17, and the VL of the PD-L1 binding domain comprises the sequence at least 90%or 95%identical to the sequence set forth in SEQ ID NO: 18.
In some embodiments, the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region. In certain embodiments, the heavy chain constant region comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the heavy chain constant region comprises a constant region of human IgG1. In certain embodiments, the IgG1 comprises one or more mutations that can confer increased CDC or ADCC relative to wild-type constant region. In certain embodiments, the one or more mutations is selected from the group consisting of S239D, I332E, H268F, S324T S236A, G236A, P247I, A339 (D/Q) , D280H, K290S, S298 (D/V) , F243L, R292P, Y300L, P396L, V305I, K290 (E/N) , S298G, T299A, K326E, E382V, M428I, S298A, K326A, E333A, K334A, S298A, E333A, and K334A, according to EU numbering. In certain embodiments, the one or more mutations comprise a combination of S298A, E333A, and K334A, according to EU numbering.
In some embodiments, the PD-L1 binding domain is a scFv (single chain fragment variable) domain. In some embodiments, the scFv domain is linked to a heavy chain
constant region. In some embodiments, the scFv domain is linked to a light chain constant region.
In some embodiments, the anti-CD4 and anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 21; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-CD4 and anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 9; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 22.
In some embodiments, the isolated antibody is a murine, a rodent, a rabbit, a chimeric, a humanized, or a human antibody.
In some embodiments, the antigen-binding fragment is a recombinant ScFv (single chain fragment variable) antibody, a Fab fragment, a F (ab’) 2 fragment, or a Fv fragment.
In some embodiments, the isolated antibody or an antigen-binding fragment thereof disclosed herein is linked to one or more conjugate moieties. In some embodiments, the conjugate moiety is an immune-modulatory agent, an anti-tumor drug, a clearance-modifying agent, a toxin, a detectable label, an RNA, a DNA, a cytokine, or purification moiety.
In another aspect, the present disclosure provides a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof disclosed herein, and a pharmaceutically acceptable carrier.
In another aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof disclosed herein.
In another aspect, the present disclosure provides a vector comprising the polynucleotide encoding the antibody or antigen-binding fragment thereof disclosed herein.
In another aspect, the present disclosure provides a host cell comprising the vector disclosed herein.
In yet another aspect, the present disclosure provides a method of producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell disclosed herein under the condition at which the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.
In another aspect, the present disclosure provides a method of treating or ameliorating the effect of a cancer in a subject. In some embodiments, the method comprises
administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof disclosed herein, or the pharmaceutical composition disclosed herein.
In some embodiments, the cancer is selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer.
In some embodiments, the subject is human.
In some embodiments, the antibody or an antigen-binding fragment thereof is administered intravenously, intra-arterially, intra-tumorally, intra-muscularly, or subcutaneously.
In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof disclosed herein in the manufacture of a medication for treating cancer in a subject.
BRIEF DESCFRIPTION OF FIGURES
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 shows the configuration of an exemplary bispecific antibody.
FIG. 2 shows the configuration of an exemplary bispecific antibody.
FIG. 3 shows binding of anti-CD4/anti-PD-L1 bispecific antibody with human CD4.
FIG. 4 shows binding of anti-CD4/anti-PD-L1 bispecific antibody with human PD-L1.
FIG. 5 shows the secretion of interleukin 2 (IL-2) induced by anti-CD4/anti-PD-L1 bispecific antibody.
FIG. 6 shows the secretion of interferon γ (IFN-γ) induced by anti-CD4/anti-PD-L1 bispecific antibody.
FIG. 7 shows antibody-dependent cell-mediated cytotoxicity (ADCC) effect of anti-CD4/anti-PD-L1 bispecific antibody.
The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.
I. Definitions
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. In this disclosure, the term “or” is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive. As used herein “another” may mean at least a second or more. Furthermore, the use of the term “including” , as well as other forms, such as “includes” and “included” , is not limiting. Also, terms such as “element” or “component” encompass both element or component comprising one unit and elements or components that comprise more than one subunit unless specifically stated otherwise. Also, the use of the term “portion” can include part of a moiety or the entire moiety.
As used herein, the singular forms “a” , “an” and “the” include plural references unless the context clearly dictates otherwise.
The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multi-specific antibody, or bispecific antibody that binds to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable domain (VH) and a constant region including a first, second, and third constant domain (CH1,
CH2, CH3, respectively) ; mammalian light chains are classified as λ or κ, while each light chain consists of a variable domain (VL) and a constant domain (CL) . A typical IgG antibody has a “Y”shape, with the stem of the Y typically consisting of the second and third constant domains of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable domain and first constant domain of a single heavy chain bound to the variable and constant domains of a single light chain. The variable domains of the light and heavy chains are responsible for antigen binding. The variable domains in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. (1985) 186 (3) : 651-63; Chothia, C. and Lesk, A. M., J. Mol. Biol. (1987) 196: 901; Chothia, C. et al., Nature (1989) 342 (6252) : 877-83; Marie-Paule Lefranc et al., Developmental and Comparative Immunology (2003) 27: 55-77; Marie-Paule Lefranc et al., Immunome Research (2005) 1 (3) ; Marie-Paule Lefranc, Molecular Biology of B cells (second edition) , chapter 26, 481-514, (2015) ) . The three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant domains of the heavy and light chains are not involved in antigen-binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (gamma1 heavy chain) , IgG2 (gamma2 heavy chain) , IgG3 (gamma3 heavy chain) , IgG4 (gamma4 heavy chain) , IgA1 (alpha1 heavy chain) , or IgA2 (alpha2 heavy chain) .
The term “antigen” refers to a substance capable of inducing adaptive immune responses. Specifically, an antigen is a substance specifically bound by antibodies or T lymphocyte antigen receptors. Antigens are usually proteins and polysaccharides, less frequently also lipids. Suitable antigens include without limitation parts of bacteria (coats, capsules, cell walls, flagella, fimbrai, and toxins) , viruses, and other microorganisms. Antigens also include tumor antigens, e.g., antigens generated by mutations in tumors. As used herein, antigens also include immunogens and haptens.
The term “antigen-binding fragment” as used herein refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a bispecific antibody, a multi-specific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody binds.
A “Fab fragment” comprises one light chain and the CH1 and variable domains of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
A “Fab′fragment” comprises one light chain and a portion of one heavy chain that contains the VH domain and the CH1 domain and also the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F (ab′) 2 molecule.
A “F (ab′) 2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F (ab′) 2 fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the two heavy chains.
“Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen-binding site. An Fv fragment consists of the variable domain of a single light chain bound to the variable domain of a single heavy chain.
“Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable domain and a heavy chain variable domain connected to one another directly or via a peptide linker sequence (Huston JS et al., Proc Natl Acad Sci USA (1988) 85: 5879) .
An “Fc” region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The Fc region of the antibody is responsible for various effector functions such as antibody-dependent cell-mediated
cytotoxicity (ADCC) , and complement dependent cytotoxicity (CDC) , but does not function in antigen binding.
“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable domain of a single light chain and the variable domain of a single heavy chain is a disulfide bond. In some embodiments, a “ (dsFv) 2” or “ (dsFv-dsFv') ” comprises three peptide chains: two VH domains linked by a peptide linker (e.g., a long flexible linker) and bound to two VL domains, respectively, via disulfide bridges. In some embodiments, dsFv-dsFv' is bispecific in which each disulfide paired heavy and light chain has a different antigen specificity.
“Camelized single domain antibody, ” “heavy chain antibody, ” or “HCAb” refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally derived from Camelidae (camels, dromedaries, and llamas) . Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature (1993) 363: 446-8; Nguyen VK. et al., Immunogenetics (2002) 54: 39-47; Nguyen VK. et al., Immunology (2003) 109: 93-101) . The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. (2007) 21: 3490-8) .
A “nanobody” refers to an antibody fragment that consists of a VHH domain from a heavy chain antibody and two constant domains, CH2 and CH3.
“Diabodies” or “dAbs” include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a VH domain connected to a VL domain in the same polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A.Jul 15; 90 (14) : 6444-8 (1993) ; EP404097; WO93/11161) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen–binding sites may target the same or different antigens (or epitopes) . In certain embodiments, a “bispecific ds diabody” is a diabody target two different antigens (or epitopes) .
In certain embodiments, an “scFv dimer” is divalent (or bivalent) single-chain variable fragments (di-scFvs, bi-scFvs) that can be engineered by linking two scFvs. A bivalent diabody or bivalent scFv (BsFv, di-scFvs, bi-scFvs) comprising VH-VL (linked by a peptide linker) dimerized with another VH-VL moiety such that VH's of one moiety coordinate with the VL's of the other moiety and form two binding sites which can target the same antigens (or epitopes) or different antigens (or epitopes) . In other embodiments, an “scFv dimer” is a bispecific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (also linked by a peptide linker) such that VH1 and VL1 coordinate and VH2 and VL2 coordinate and each coordinated pair has a different antigen specificity.
A “domain antibody” refers to an antibody fragment containing only the variable domain of a heavy chain or the variable domain of a light chain. In certain instances, two or more VH domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.
A “bispecific” antibody refers to an artificial antibody which has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes. The two epitopes may present on the same antigen, or they may present on two different antigens.
“Cancer” as used herein refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies) such as leukemia. As used herein “solid tumor” refers to a solid mass of neoplastic and/or malignant cells. Examples of cancer or tumors include hematological malignancies, oral carcinomas (for example of the lip, tongue or pharynx) , digestive organs (for example esophagus, stomach, small intestine, colon, large intestine, or rectum) , peritoneum, liver and biliary passages, pancreas, respiratory system such as larynx or lung (small cell and non-small cell) , bone, connective tissue, skin (e.g., melanoma) , breast, reproductive organs (fallopian tube, uterus, cervix, testicles, ovary, or prostate) , urinary tract (e.g., bladder or kidney) , brain and endocrine glands such as the thyroid. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, renal cancer, bladder cancer, hepatocellular cancer, and colorectal cancer. In certain embodiments, the cancer is selected from a lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma.
The term “chimeric” as used herein, means an antibody or antigen-binding fragment, having a portion of heavy and/or light chain derived from one species, and the rest of the heavy and/or light chain derived from a different species. In an illustrative example, a chimeric antibody may comprise a constant region derived from human and a variable region from a non-human animal, such as from mouse or rabbit. In some embodiments, the non-human animal is a mammal, for example, a mouse, a rat, a rabbit, a goat, a sheep, a guinea pig, or a hamster.
The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human CD4 or PD-L1 with a binding affinity (KD) of ≤10-6 M (e.g., ≤5x10-7 M, ≤2x10-7 M, ≤10-7 M, ≤5x10-8 M, ≤2x10-8 M, ≤10-8 M, ≤5x10-9 M, ≤4x10-9M, ≤3x10-
9M, ≤2x10-9 M, or ≤10-9 M) . KD used herein refers to the ratio of the dissociation rate to the association rate (koff/kon) , which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. In certain embodiments, the KD value can be appropriately determined by using flow cytometry.
The ability to “block binding” or to “compete for the same epitope” as used herein refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human CD4 and an anti-CD4 antibody) to any detectable degree. In certain embodiments, an antibody or antigen-binding fragment that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 85%, or greater than 90%.
Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a given antibody binds to the same epitope as the antibody of present disclosure by ascertaining whether the former prevents the latter from binding to a CD4 antigen polypeptide. If the given antibody competes with the antibody of present disclosure, as shown by a decrease in binding by the antibody of present disclosure to the CD4 antigen polypeptide, then the two antibodies bind to the same, or a closely related, epitope. Or if the binding of a given antibody to the CD4 antigen polypeptide was inhibited by the antibody of present disclosure, then the two antibodies bind to the same, or a closely related, epitope.
“CD4” as used herein, refers to CD4 derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats) . Exemplary sequence of human CD4 includes GenBank SEQ Reference No. NP_000607, NP_001181943, NP_001181944, NP_001181945, and NP_001181946. The term “CD4” as used herein is intended to encompass any form of human CD4, for example, 1) native unprocessed CD4 molecule, “full-length” CD4 chain or naturally occurring variants of CD4, including, for example, splice variants or allelic variants; 2) any form of CD4 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of CD4 subunit generated through recombinant method.
“PD-L1” as used herein, refers to PD-L1 derived from any vertebrate source, including mammals such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats) . Exemplary sequence of human PD-L1 includes GenBank SEQ Reference No. NP_001254635, NP_001300958, and NP_054862. The term “PD-L1” as used herein is intended to encompass any form of human PD-L1, for example, 1) native unprocessed PD-L1 molecule, “full-length” PD-L1 chain or naturally occurring variants of PD-L1, including, for example, splice variants or allelic variants; 2) any form of PD-L1 that results from processing in the cell; or 3) full length, a fragment (e.g., a truncated form, an extracellular/transmembrane domain) or a modified form (e.g. a mutated form, a glycosylated/PEGylated, a His-tag/immunofluorescence fused form) of PD-L1 subunit generated through recombinant method.
The term “anti-CD4 anti-PD-L1 bispecific antibody” refers to an antibody that is capable of simultaneously specifically binding to CD4 (e.g., human CD4) and PD-L1 (e.g., human PD-L1) .
A “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g., Asp, Glu) , among amino acids with basic side chains (e.g., His, Lys, and Arg) , or among residues with aromatic side chains (e.g., Trp, Tyr, and Phe) . As known in the art, conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
“Effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as C1 complex and Fc receptor. Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and phagocytosis.
The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same or a closely related epitope within an antigen if they exhibit competitive binding for the antigen. For example, if an antibody or antigen-binding fragment blocks binding of a reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding fragment may be considered to bind the same/closely related epitope as the reference antibody.
The term “homologue” and “homologous” as used herein are interchangeable and refer to nucleic acid sequences (or its complementary strand) or amino acid sequences that have sequence identity of at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequence when optimally aligned.
The phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
The term “humanized” as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, the constant regions derived from human.
An “isolated” substance has been altered by the hand of man from the natural state. If an “isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated, ” but the same polynucleotide or polypeptide is “isolated” if it has been sufficiently separated from the coexisting materials of its natural state so as to exist in a substantially pure state. An “isolated nucleic acid sequence” refers to the sequence of an isolated nucleic acid molecule. In certain embodiments, an “isolated antibody or antigen-binding fragment thereof” refers to the antibody or antigen-binding fragments having a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%as determined by
electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) , or chromatographic methods (such as ion exchange chromatography or reverse phase HPLC) .
A “leader peptide” or “signal peptide” refers to a peptide having a length of about 5-30 amino acids that is present at the N-terminus of newly synthesized proteins that form part of the secretory pathway. Proteins of the secretory pathway include, but are not limited to proteins that reside either inside certain organelles (the endoplasmic reticulum, Golgi or endosomes) , are secreted from the cell, or are inserted into a cellular membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of a protein.
The term “link” as used herein refers to the association via intramolecular interaction, e.g., covalent bonds, metallic bonds, and/or ionic bonding, or inter-molecular interaction, e.g., hydrogen bond or noncovalent bonds.
The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given signal peptide that is operably linked to a polypeptide directs the secretion of the polypeptide from a cell. In the case of a promoter, a promoter that is operably linked to a coding sequence will direct the expression of the coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.
“Percent (%) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) . Conservative substitution of the amino acid residues may or may not be considered as identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S. F. et al., J. Mol. Biol. (1990) 215: 403–410; Stephen F. et al., Nucleic Acids Res. (1997) 25: 3389–3402) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D. G. et al., Methods in Enzymology (1996) 266: 383-402; Larkin M. A. et al., Bioinformatics (2007) 23: 2947-8) , and ALIGN or Megalign (DNASTAR) software. Those
skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm.
The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2′, 3′-dideoxyribose, and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate.
The term “polypeptide” or “protein” means a string of at least two amino acids linked to one another by peptide bonds. Polypeptides and proteins may include moieties in addition to amino acids (e.g., may be glycosylated) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “polypeptide” or “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a polypeptide or protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. The term also includes amino acid polymers in which one or more amino acids are chemical analogs of a corresponding naturally-occurring amino acid and polymers.
The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) . A human includes pre-and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient. ” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
The term “therapeutically effective amount” or “effective dosage” as used herein refers to the dosage or concentration of a drug effective to treat a disease or condition.
For example, with regard to the use of the monoclonal antibodies or antigen-binding fragments thereof disclosed herein to treat cancer, a therapeutically effective amount is the dosage or concentration of the monoclonal antibody or antigen-binding fragment thereof capable of reducing the tumor volume, eradicating all or part of a tumor, inhibiting or slowing tumor growth or cancer cell infiltration into other organs, inhibiting growth or proliferation of cells mediating a cancerous condition, inhibiting or slowing tumor cell metastasis, ameliorating any symptom or marker associated with a tumor or cancerous condition, preventing or delaying the development of a tumor or cancerous condition, or some combination thereof.
“Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
The term “vector” as used herein refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein. A vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, and papovavirus (e.g., SV40) . A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication. A vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating. A vector can be an expression vector or a cloning vector. The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP,
pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT. RTM., pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
II. Anti-CD4 Anti-PD-L1 Bispecific Antibody and Antigen-binding Fragment
The present disclosure in one aspect provides an anti-CD4 anti-PD-L1 bispecific antibody and antigen-binding fragment thereof that has a high binding affinity to both human CD4 and human PD-L1. In certain embodiments, the bispecific antibody or antigen-binding fragment, when bound to PD-L1, can specifically interfere with, block or reduce the interaction between PD-L1 and its receptor PD-1. In certain embodiments, the bispecific antibodies or antigen-binding fragments, when bound to CD4, induces CDC or ADCC.
Binding affinity of the antibody and antigen-binding fragment provided herein can be represented by KD value, which represents the ratio of dissociation rate to association rate (koff/kon) when the binding between the antigen and antigen-binding molecule reaches equilibrium. The antigen-binding affinity (e.g., KD) can be appropriately determined using suitable methods known in the art, including, for example, bio-layer interferometry.
Binding of the antibodies to human CD4 or PD-L1 can also be represented by “half maximal effective concentration” (EC50) value, which refers to the concentration of an antibody where 50%of its maximal effect (e.g., binding or inhibition etc. ) is observed. The EC50 value can be measured by methods known in the art, for example, sandwich assay such as ELISA, Western Blot, flow cytometry assay, and other binding assays.
CD4 and PD-L1 Binding Domains
The anti-CD4 anti-PD-L1 bispecific antibody provided herein comprises a CD4 binding domain and a PD-L1 binding domain. Each of the CD4 binding domain and PD-L1 binding domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of an anti-CD4 or anti-PD-L1 antibody disclosed herein. CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are indispensable or
unchangeable. In other words, it is possible to replace or change or modify one or more CDRs disclosed herein, yet substantially retain the specific binding affinity to CD4 or PD-L1.
In certain embodiments, the CD4 binding domain and PD-L1 binding domain have a CDR sequence as listed in Table 1 below.
Table 1. CDR sequences of the CD4 and PD-L1 binding domains
In certain embodiments, the CD4 binding domain and PD-L1 binding domain provided herein comprise suitable framework region (FR) sequences, as long as the domain can specifically bind to CD4 or PD-L1. The CDR sequences provided in Table 1 can be grafted to any suitable FR sequences of any suitable species such as mouse, human, rat, rabbit, among others, using suitable methods known in the art such as recombinant techniques.
In certain embodiments, the CD4 binding domain and PD-L1 binding domain provided herein are humanized. A humanized binding domain is desirable in its reduced immunogenicity in human. A humanized domain is chimeric, as non-human CDR sequences are grafted to human or substantially human FR sequences. Humanization of a binding domain can be essentially performed by substituting the non-human (such as murine) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al., Nature (1986) 321: 522-525; Riechmann et al., Nature (1988) 332: 323-327; Verhoeyen et al., Science (1988) 239: 1534-1536) .
Suitable human heavy chain and light chain variable domains can be selected to achieve this purpose using methods known in the art. In an illustrative example, “best-fit” approach can be used, where a non-human (e.g., rodent) antibody variable domain sequence is
screened or BLASTed against a database of known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as the human scaffold for grafting the non-human CDR sequences (see, for example, Sims et al., J. Immunol. (1993) 151: 2296; Chothia et al., J. Mot. Biol. (1987) 196: 901) . Alternatively, a framework derived from the consensus sequence of all human antibodies may be used for the grafting of the non-human CDRs (see, for example, Carter et at. Proc. Natl. Acad. Sci. USA (1992) 89:4285; Presta et al., J. Immunol. (1993) 151: 2623) .
In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences except for the CDR sequences which are non-human. In some embodiments, the variable region FRs, and constant regions if present, are entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences may be derived different human immunoglobulin genes, for example, FR sequences derived from one human antibody and constant region from another human antibody.
In certain embodiments, the CD4 binding domain and PD-L1 binding domain provided herein comprise paired heavy chain and light chain variable region amino acid sequences as provided in Table 2 below.
Table 2. Clone-Paired VH and VL Sequences
In some embodiments, the CD4 binding domain or PD-L1 binding domain is a single chain variable fragment (svFv) . A scFv is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. In certain embodiments, the linker comprises an amino acid sequence of SEQ ID NO.
19.This chimeric molecule retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered. These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A/G) used to purify antibodies. These fragments can often be purified/immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.
Flexible linkers generally are comprised of helix-and turn-promoting amino acid residues such as alanine, serine and glycine. However, other residues can function as well. Tang et al. (1996) used phage display as a means of rapidly selecting tailored linkers for single-chain antibodies (scFvs) from protein linker libraries. A random linker library was constructed in which the genes for the heavy and light chain variable domains were linked by a segment encoding an 18-amino acid polypeptide of variable composition. The scFv repertoire (approx. 5 × 106 different members) was displayed on filamentous phage and subjected to affinity selection with hapten. The population of selected variants exhibited significant increases in binding activity but retained considerable sequence diversity. Screening 1054 individual variants subsequently yielded a catalytically active scFv that was produced efficiently in soluble form. Sequence analysis revealed a conserved proline in the linker two residues after the VH C terminus and an abundance of arginines and prolines at other positions as the only common features of the selected tethers.
Configuration of the Bispecific Antibody
The bispecific antibodies and antigen-binding fragments thereof provided herein can be in a suitable format known in the art. For example, an exemplary bispecific format can be IgG-scFv fusions, dual variable domain (DVD) -Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc. ) , BiTE, CrossMab, CrossFab, Duobody, SEEDbody, leucine zipper, dual acting Fab (DAF) -IgG, and Mab2 bispecific formats, bispecific diabodies, scFv-based bispecific formats (see, e.g., Brinkmann et al.2017, Mabs, 9 (2) : 182-212) . The bispecific molecules can be in symmetric or asymmetric architecture.
In certain embodiments, the bispecific antibodies and the fragments thereof provided herein further comprise an immunoglobulin constant region. In some embodiments,
an immunoglobulin constant region comprises a heavy chain and/or a light chain constant region. The heavy chain constant region comprises CH1, hinge, and/or CH2-CH3 regions. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises Cκ or Cλ.
In some embodiments, the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region. In certain embodiments, the heavy chain constant region comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the heavy chain constant region comprises a constant region of human IgG1.
In some embodiments, the PD-L1 binding domain is a scFv (single chain fragment variable) domain. In some embodiments, the scFv domain is linked to a heavy chain constant region. In some embodiments, the scFv domain is linked to a light chain constant region.
In some embodiments, the anti-CD4 anti-PD-L1 bispecific antibody has a configuration illustrated in FIG. 1. As shown in FIG. 1, the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region. The PD-L1 binding domain is a scFv domain linked to a heavy chain constant region.
In some embodiments, the anti-CD4 anti-PD-L1 bispecific antibody has a configuration illustrated in FIG. 2. As shown in FIG. 2, the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region. The PD-L1 binding domain is a scFv domain linked to a light chain constant region.
In some embodiments, the anti-CD4 anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 21; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-CD4 and anti-PD-L1 bispecific antibody comprises (i) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 9; and (ii) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 22.
The bispecific antibodies and antigen-binding fragments provided herein can be made with any suitable methods known in the art. In one embodiment, two immunoglobulin heavy chain-light chain pairs having different antigenic specificities are co-expressed in a host
cell to produce bispecific antibodies in a recombinant way (see, for example, Milstein and Cuello, Nature, 305: 537 (1983) ) , followed by purification by affinity chromatography.
Antibody Variants
The antibodies and antigen-binding fragments thereof provided herein also encompass various variants thereof. In certain embodiments, the antibodies and antigen-binding fragments thereof encompasses various types of variants of an exemplary antibody provided herein.
In certain embodiments, the antibody variants comprise one or more modifications or substitutions in one or more CDR sequences as provided in Table 1, one or more variable region sequences (but not in any of the CDR sequences) provided herein, and/or the constant region (e.g., Fc region) . Such variants retain specific binding affinity to CD4 or PD-L1 of their parent antibodies, but have one or more desirable properties conferred by the modification (s) or substitution (s) . For example, the antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamidation or deamination, improved or increased effector function (s) , reduced or depleted effector function (s) , improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and/or compatibility to conjugation (e.g., one or more introduced cysteine residues) .
The parent antibody sequence may be screened to identify suitable or preferred residues to be modified or substituted, using methods known in the art, for example “alanine scanning mutagenesis” (see, for example, Cunningham and Wells (1989) Science, 244: 1081-1085) . Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) , and the modified antibodies are produced and screened for the interested property. If substitution at a particular amino acid location demonstrates an interested functional change, then the position can be identified as a potential residue for modification or substitution. The potential residues may be further assessed by substituting with a different type of residue (e.g. cysteine residue, positively charged residue, etc. ) .
Affinity variant
Affinity variant may contain modifications or substitutions in one or more CDR sequences, one or more FR sequences, or the heavy or light chain variable region sequences provided herein. The affinity variants retain specific binding affinity to CD4 or PD-L1 of the
parent antibody, or even have improved CD4 or PD-L1 specific binding affinity over the parent antibody.
Various methods known in the art can be used to achieve this purpose. For example, a library of antibody variants (such as Fab or scFv variants) can be generated and expressed with phage display technology, and then screened for the binding affinity to human CD4 or PD-L1. For another example, computer software can be used to virtually simulate the binding of the antibodies to human CD4 or PD-L1 and identify the amino acid residues on the antibodies which form the binding interface. Such residues may be either avoided in the substitution so as to prevent reduction in binding affinity or targeted for substitution to provide for a stronger binding.
In certain embodiments, the humanized antibody or antigen-binding fragment provided herein comprises one or more amino acid residue substitutions in one or more CDR sequences, and/or one or more FR sequences. In certain embodiments, an affinity variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitution in the CDR sequences and/or FR sequences in total.
In certain embodiments, the bispecific antibodies and antigen-binding fragments thereof comprise 1, 2, or 3 CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) listed in Table 1, and in the meantime retain the binding affinity to CD4 and PD-L1 at a level similar to or even higher than its parent antibody.
In certain embodiments, the bispecific antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to that (or those) provided herein, and in the meantime retain the binding affinity to CD4 and PD-L1 at a level similar to or even higher than its parent antibody. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) .
Glycosylation variant
In still another embodiment, the antibody comprises a particular glycosylation pattern. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation) . The glycosylation pattern of an antibody may be altered to, for example, increase the affinity or avidity of the antibody for an antigen. Such modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the
antibody sequence. For example, one or more amino acid substitutions can be made that result removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity or avidity of the antibody for antigen. See, e.g., U.S. Patents 5,714,350 and 6,350,861.
An antibody may also be made in which the glycosylation pattern includes hypofucosylated or afucosylated glycans, such as a hypofucosylated antibodies or afucosylated antibodies have reduced amounts of fucosyl residues on the glycan. The antibodies may also include glycans having an increased amount of bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such modifications can be accomplished by, for example, expressing the antibodies in a host cell in which the glycosylation pathway was been genetically engineered to produce glycoproteins with particular glycosylation patterns. These cells have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α (1, 6) -fucosyltransferase) , such that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8-/-cell lines were created by the targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S. Patent Publication No. 20040110704. As another example, EP 1 176 195 describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation by reducing or eliminating the α-1, 6 bond-related enzyme. EP 1 176 195 also describes cell lines which have a low enzyme activity for adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activity, for example the rat myeloma cell line YB2/0 (ATCC CRL 1662) . PCT Publication WO 2003/035835 describes a variant CHO cell line, Lec13 cells, with reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell. Antibodies with a modified glycosylation profile can also be produced in chicken eggs, as described in PCT Publication WO 06/089231. Alternatively, antibodies with a modified glycosylation profile can be produced in plant cells, such as Lemna (US Patent 7, 632, 983) . Methods for production of antibodies in a plant system are disclosed in the U.S. Patents 6,998,267 and 7,388,081. PCT Publication WO1999/054342 describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., β (1, 4) -N-acetylglucosaminyltransferase III (GnTIII) ) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac
structures which results in increased ADCC activity of the antibodies. Hypofucosylation is also called afucosylation when fucosylation is minimal on antibodies.
Alternatively, the fucose residues of the antibodies can be cleaved off using a fucosidase enzyme; e.g., the fucosidase α-L-fucosidase removes fucosyl residues from antibodies. Antibodies disclosed herein further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian-or human-like glycosylation patterns. A particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Patents 7,029,872 and 7,449,308) . These genetically modified host cells have been used to produce antibodies that have predominantly particular N-glycan structures.
In addition, since fungi such as yeast or filamentous fungi lack the ability to produce fucosylated glycoproteins, antibodies produced in such cells will lack fucose unless the cells are further modified to include the enzymatic pathway for producing fucosylated glycoproteins (See for example, PCT Publication WO2008112092) . In particular embodiments, the antibodies disclosed herein further include those produced in lower eukaryotic host cells and which comprise fucosylated and nonfucosylated hybrid and complex N-glycans, including bisected and multiantennary species, including but not limited to N-glycans such as GlcNAc (1-4)Man3GlcNAc2; Gal (1-4) GlcNAc (1-4) Man3GlcNAc2; NANA (1-4) Gal (1-4) GlcNAc (1-4)Man3GlcNAc2. In particular embodiments, the antibody compositions provided herein may comprise antibodies having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2; GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In particular aspects, the hybrid N-glycan is the predominant N-glycan species in the composition. In further aspects, the hybrid N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%of the hybrid N-glycans in the composition.
In particular embodiments, the antibody compositions provided herein comprise antibodies having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2; GalGlcNAcMan3GlcNAc2; NANAGalGlcNAcMan3GlcNAc2; GlcNAc2Man3GlcNAc2; GalGlcNAc2Man3GlcNAc2; Gal2GlcNAc2Man3GlcNAc2; NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In particular
aspects, the complex N-glycan is the predominant N-glycan species in the composition. In further aspects, the complex N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%of the complex N-glycans in the composition. In particular embodiments, the N-glycan is fusosylated. In general, the fucose is in an α1, 3-linkage with the GlcNAc at the reducing end of the N-glycan, an α1, 6-linkage with the GlcNAc at the reducing end of the N-glycan, an α1, 2-linkage with the Gal at the non-reducing end of the N-glycan, an α1, 3-linkage with the GlcNac at the non-reducing end of the N-glycan, or an α1, 4-linkage with a GlcNAc at the non-reducing end of the N-glycan.
Therefore, in particular aspects of the above the glycoprotein compositions, the glycoform is in an α1, 3-linkage or α1, 6-linkage fucose to produce a glycoform selected from the group consisting of Man5GlcNAc2 (Fuc) , GlcNAcMan5GlcNAc2 (Fuc) , Man3GlcNAc2 (Fuc) , GlcNAcMan3GlcNAc2 (Fuc) , GlcNAc2Man3GlcNAc2 (Fuc) , GalGlcNAc2Man3GlcNAc2 (Fuc) , Gal2GlcNAc2Man3GlcNAc2 (Fuc) , NANAGal2GlcNAc2Man3GlcNAc2 (Fuc) , and NANA2Gal2GlcNAc2Man3GlcNAc2 (Fuc) ; in an α1, 3-linkage or α1, 4-linkage fucose to produce a glycoform selected from the group consisting of GlcNAc (Fuc) Man5GlcNAc2, GlcNAc (Fuc) Man3GlcNAc2, GlcNAc2 (Fuc1-2)Man3GlcNAc2, GalGlcNAc2 (Fuc1-2) Man3GlcNAc2, Gal2GlcNAc2 (Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2 (Fuc1-2) Man3GlcNAc2, and NANA2Gal2GlcNAc2 (Fuc1-2) Man3GlcNAc2; or in an α1, 2-linkage fucose to produce a glycoform selected from the group consisting of Gal (Fuc) GlcNAc2Man3GlcNAc2, Gal2 (Fuc1-2) GlcNAc2Man3GlcNAc2, NANAGal2 (Fuc1-2) GlcNAc2Man3GlcNAc2, and NANA2Gal2 (Fuc1-2) GlcNAc2Man3GlcNAc2.
In further aspects, the antibodies comprise high mannose N-glycans, including but not limited to, Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or N-glycans that consist of the Man3GlcNAc2 N-glycan structure. In further aspects of the above, the complex N-glycans further include fucosylated and non-fucosylated (or afucosylated) bisected and multiantennary species. As used herein, the terms "N-glycan" and "glycoform" are used interchangeably and refer to an N-linked oligosaccharide, for example, one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.
The bispecific antibodies and antigen-binding fragments provided herein also encompass a glycosylation variant, which can be obtained to either increase or decrease the extent of glycosylation of the antibody or antigen binding fragment.
The antibody or antigen binding fragment thereof may comprise one or more amino acid residues with a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, for example, an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) or serine or threonine residues (for O-linked glycosylation sites) present in the sequence in the is substituted. A new glycosylation site can be created in a similar way by introducing such a tripeptide sequence or serine or threonine residue.
Cysteine-engineered variant
The anti-CD4 and anti-PD-L1 bispecific antibodies and antigen-binding fragments provided herein also encompass a cysteine-engineered variant, which comprises one or more introduced free cysteine amino acid residues.
A free cysteine residue is one which is not part of a disulfide bridge. A cysteine-engineered variant is useful for conjugation with for example, a cytotoxic and/or imaging compound, a label, or a radioisoptype among others, at the site of the engineered cysteine, through for example a maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
Fc Variant
The antibodies disclosed herein can also be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity) . Furthermore, the antibodies disclosed herein can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or
be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat. The antibodies disclosed herein also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Patent 5,624,821; WO2003/086310; US2004/0002587; US2005/0152894; US2005/0249723; WO2006/019447. Such modification can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions) , glycosylation or deglycosylation, and adding multiple Fc. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region.
In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Patent 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patents 5,869,046 and 6,121,022. In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function (s) of the antibodies. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patents 5,624,821 and 5,648,260.
In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO1994/029351. In yet another example, the Fc region is modified to increase or decrease the ability of the antibodies to
mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase or decrease the affinity of the antibodies for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 2000/042072. Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described. Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcγRIII. Additionally, the following combination mutants were shown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A.
In one embodiment, the Fc region is modified to decrease the ability of the antibodies to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.
In one embodiment, the Fc region is modified to abolish the ability of the antibodies to mediate effector function by modifying residues 234, 235 and 329 to alanine or glycine (L234A-L235A-P329G) .
The bispecific antibodies and antigen-binding fragments provided herein also encompass an Fc variant, which comprises one or more amino acid residue modifications or substitutions at its Fc region and/or hinge region.
In certain embodiments, the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) that improves pH-dependent binding to neonatal Fc receptor (FcRn) . Such a variant can have an extended pharmacokinetic half-life, as it binds to FcRn at acidic pH which allows it to escape from degradation in the lysosome and then be translocated and released out of the cell. Methods of engineering an antibody and antigen-binding fragment thereof to improve binding affinity with FcRn are well-known in the art, see, for example, Vaughn, D. et al., Structure, 6 (1) : 63-73, 1998; Kontermann, R.et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for
improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research (2010) 70: 3269-3277; and Hinton, P. et al., J. Immunology (2006) 176: 346-356.
In certain embodiments, the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) that alters the antibody-dependent cellular cytotoxicity (ADCC) . Certain amino acid residues at CH2 domain of the Fc region can be substituted to provide for enhanced ADCC activity. Alternatively, or additionally, carbohydrate structures on the antibody can be changed to enhance ADCC activity. Methods of altering ADCC activity by antibody engineering have been described in the art, see for example, Shields RL. et al., J Biol Chem. (2001) 276 (9) : 6591-604; Idusogie EE. et al., J Immunol. (2000) 164 (8) : 4178-84; Steurer W. et al., J Immunol. (1995) 155 (3) : 1165-74; Idusogie EE. et al., J Immunol. (2001) 166 (4) : 2571-5; Lazar GA. et al., PNAS (2006) 103 (11) : 4005-4010; Ryan MC. et al., Mol. Cancer Ther. (2007) 6: 3009-3018; Richards JO. et al., Mol Cancer Ther. (2008) 7 (8) : 2517-27; Shields R. L. et al., J. Biol. Chem, 2002, 277: 26733-26740; Shinkawa T. et al., J. Biol. Chem (2003) 278: 3466-3473.
In certain embodiments, the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) that alters Complement Dependent Cytotoxicity (CDC) , for example, by improving or diminishing C1q binding and/or CDC (see, for example, WO99/51642; Duncan &Winter Nature 322: 738-40 (1988) ; U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821) ; and WO1994/029351 concerning other examples of Fe region variants.
In certain embodiments, the bispecific antibodies or antigen-binding fragments disclosed herein comprise one or more amino acid substitution (s) in the interface of the Fc region to facilitate and/or promote heterodimerization. These modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance can be positioned in the cavity so as to promote interaction of the first and second Fc polypeptides to form a heterodimer or a complex. Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731,168.
Antigen-binding fragments
Provided herein are also anti-CD4 anti-PD-L1 bispecific antigen-binding fragments. Various types of antigen-binding fragments are known in the art and can be developed based on the bispecific antibodies provided herein, including for example, the exemplary antibodies whose CDR and variable sequences are provided herein, and their
different variants (such as affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants and so on) .
In certain embodiments, an anti-CD4 anti-PD-L1 bispecific antigen-binding fragment provided herein is a camelized single domain antibody, a diabody, a single chain Fv fragment (scFv) , an scFv dimer, a BsFv, a dsFv, a (dsFv) 2, a dsFv-dsFv', an Fv fragment, a Fab, a Fab', a F (ab') 2, a bispecific antibody, a ds diabody, a nanobody, a domain antibody, a single domain antibody, or a bivalent domain antibody.
Conjugates
In some embodiments, the bispecific antibodies and antigen-binding fragments thereof further comprise a conjugate moiety. The conjugate moiety can be linked to the antibodies and antigen-binding fragments thereof. A conjugate moiety is a proteinaceous or non-proteinaceous moiety that can be attached to the antibody or antigen-binding fragment thereof. It is contemplated that a variety of conjugate moieties may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, “Conjugate Vaccines” , Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr. (eds. ) , Carger Press, New York, (1989) ) . These conjugate moieties may be linked to the antibodies or antigen-binding fragments by covalent binding, affinity binding, intercalation, coordinate binding, complexation, association, blending, or addition, among other methods.
In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain specific sites outside the epitope binding portion that may be utilized for binding to one or more conjugate moieties. For example, such a site may include one or more reactive amino acid residues, such as for example cysteine or histidine residues, to facilitate covalent linkage to a conjugate moiety.
In certain embodiments, the antibodies may be linked to a conjugate moiety indirectly, or through another conjugate moiety. For example, the antibody or antigen-binding fragments may be conjugated to biotin, then indirectly conjugated to a second conjugate that is conjugated to avidin.
Examples of conjugate moiety include without a limitation an immune-modulatory agent, an anti-tumor drug, a STING (Stimulator of Interferon Genes) agonist, a cytokine, a clearance-modifying agent, a toxin (e.g., a chemotherapeutic agent) , an immune cell stimulator (e.g., a TLR agonist) , a detectable label (e.g., a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label) , a DNA, an RNA, or
purification moiety.
Examples of immune modulatory agent include without limitation an immune modulator molecule disclosed herein (e.g., PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, Fc receptors, FCRL (1-6) , A2AR, CD160, 2B4, TGF-β, TGF-βR, VISTA, BTLA, TIGIT, LAIR1, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LILRA (1-6) , OX40, CD2, CD27, CD28, CD30, CD40, CD47, SIRPA, CLEC-1, clever-1/stabilin-1, ADGRE, TREM1, TREM2, CD122, ICAM-1, IDO, NKG2D/C, SLAMF7, MS4A4A, SIGLEC (7-15) , NKp80, NKG2A, CD160, CD161, CD300, CD163, B7-H3, LFA-1, ICOS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, TNFR2, TLR (1-9) , IL-2, IL-7, IL-15, IL-21, CD16 and CD83) , or a functional fragment thereof, a ligand thereof, and a ligand-binding protein thereof.
In some embodiments, the immune modulatory agent linked to the antibodies and antigen-binding fragments disclosed herein is a ligand-binding protein (e.g., ligand trapper) specific to an immune modulatory receptor.
Examples of anti-tumor drugs include without limitation a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenesis agent, a cancer immunotherapeutic agent, a apoptotic agent, an anti-tubulin agent, an anti-HER-2 antibody, an anti-CD20 antibody, an epidermal growth factor receptor (EGFR) antagonist, HER1/EGFR inhibitor, a platelet derived growth factor inhibitor, a COX-2 inhibitor, an interferon, a CTLA4 inhibitor (e.g., anti-CTLA antibody ipilimumabor tremelimumab) , a PD-l or PD-L1 inhibitor (e.g., or nivolumab, or pembrolizumab, or atezolizumab, or avelumab, or durvalumab, or cemiplimab-rwlc, or sintilimab, tislelizumab (BGB-A317) , penpulimab (AK105) , camrelizumab, toripalimab, zimberelimab (GLS-010) , retifanlimab, sugemalimab, or CS1003) , a dual-targeting antibody against CTLA-4 and PD-1 or PD-L1 (e.g., an anti-PD-1/CTLA-4 bi-specific antibody or AK104) , a TIM3 inhibitor (e.g., anti-TIM3 antibodies) , a LAG-3 inhibitor (e.g., anti-LAG3 antibodies) , a cytokine, an antagonist (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, FGFR2b, PDGFR-beta, BlyS, APRIL, BCMA, or VEGF receptor (s) , TRAIL/Apo2, an IDH1 inhibitor, an ivosidenib, an IDH2 inhibitor, an enasidenib, asmoothened (SMO) inhibitor, a glasdegib, an arginase inhibitor, an IDO inhibitor, an epacadostat, a BCL-2 inhibitor, a venetoclax, aplatinum complex derivative, oxaliplatin, a kinase inhibitor, a tyrosine kinase inhibitor, a PI3 kinase inhibitor, a BTK inhibitor, an ibrutinib, an acalabrutinib, azanubrutinib, a
TLR agonist, a STING agonist, an ICOS antibody, a TIGIT antibody, a CD40 antibody, a 4-1BB antibody, a CD47 antibody, an OX40 antibody, a TNFR2 antibody, an antibody to another LILR family member, a Siglec antibody, a SIRP1α antibody or fusions protein, an antagonist of E-selectin, an antibody binding to a tumor antigen, an antibody binding to a T cell surface marker, an antibody binding to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy medicine, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor, an engineered T cell, NK cell or macrophage.
A “toxin” can be any agent that is detrimental to cells or that can damage or kill cells. Examples of toxin include, without limitation, taxol, cytochalasin B, deruxtecan, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF) , mertansine, emtansine, DM1, maytansinoid DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU) , cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin) , anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin) , antibiotics (e.g., dactinomycin (formerly actinomycin) , bleomycin, mithramycin, and anthramycin (AMC) ) , anti-mitotic agents (e.g., vincristine and vinblastine) , a topoisomerase inhibitor, and a tubulin-binders.
Examples of detectable label may include a fluorescent labels (e.g. fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red) , enzyme-substrate labels (e.g. horseradish peroxidase, alkaline phosphatase, luceriferases, glucoamylase, lysozyme, saccharide oxidases or β-D-galactosidase) , radioisotopes (e.g. 123I, 124I, 125I, 131I, 35S, 3H, 111In, 112In, 14C, 64Cu, 67Cu, 86Y, 88Y, 90Y, 177Lu, 211At, 186Re, 188Re, 153Sm, 212Bi, and 32P, other lanthanides) , luminescent labels, chromophoric moiety, digoxigenin, biotin/avidin, a DNA molecule or gold for detection.
In certain embodiments, the conjugate moiety can be a clearance-modifying agent which helps increase half-life of the antibody. Illustrative examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, copolymers of ethylene glycol/propylene glycol, and the like. The polymer may
be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymers are attached, they can be the same or different molecules.
In certain embodiments, the conjugate moiety can be a purification moiety such as a magnetic bead.
In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein is used for a base for a conjugate.
Polynucleotides and Recombinant Methods
The present disclosure provides isolated polynucleotides that encode the bispecific antibodies and antigen-binding fragments thereof disclosed herein. In certain embodiments, the isolated polynucleotides comprise one or more nucleotide sequences listed in Table 1 that encodes the variable region of the exemplary antibodies provided herein. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) . The encoding DNA may also be obtained by synthetic methods.
The isolated polynucleotide that encodes the bispecific antibodies and antigen-binding fragments can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α) , and a transcription termination sequence.
The present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the antibodies or antigen-binding fragments, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker. Examples of vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT. RTM.,
pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
Vectors comprising the polynucleotide sequence encoding the antibody or antigen-binding fragment can be introduced to a host cell for cloning or gene expression. Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for bispecific antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424) , K. bulgaricus (ATCC 16,045) , K. wickeramii (ATCC 24,178) , K. waltii (ATCC 56,500) , K. drosophilarum (ATCC 36,906) , K. thermotolerans, and K. marxianus; yarrowia (EP 402,226) ; Pichia pastoris (EP 183,070) ; Candida; Trichoderma reesia (EP 244,234) ; Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
Suitable host cells for the expression of glycosylated antibodies or antigen-fragment provided here are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti (mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruit fly) , and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651) ; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. (1977) 36: 59) ; baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese Hamster Ovary cells (CHO) , CHO cells deficient in dihydrofolate reductase (DHFR) activity, CHO-DHFR (Urlaub et al., Proc. Natl. Acad. Sci. USA (1980) 77: 4216) ; mouse sertoli cells (TM4, Mather, Biol. Reprod. (1980) 23: 243-251) ; monkey kidney cells (CV1 ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34) ; buffalo rat liver cells (BRL 3A, ATCC CRL 1442) ; human lung cells (W138, ATCC CCL 75) ; human liver cells (Hep G2, HB 8065) ; mouse mammary tumor (MMT 060562, ATCC CCL51) ; TRI cells (Mather et al., Annals N. Y. Acad. Sci. (1982) 383: 44-68) ; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) . In some preferable embodiments, the host cell is 293F cell.
Host cells are transformed with the above-described expression or cloning vectors for bispecific antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In another embodiment, the antibody may be produced by homologous recombination known in the art.
The host cells used to produce the antibodies or antigen-binding fragments provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979) , Barnes et al., Anal. Biochem. (1980) 102: 255, U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCINTM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those
skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology (1992) 10: 163-167 describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
The bispecific antibodies and antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.
In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of the antibody and antigen-binding fragment thereof. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human gamma1, gamma2, or gamma4 heavy chains (Lindmark et al., J. Immunol. Meth. (1983) 62: 1-13) . Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. (1986) 5: 1567-75) . The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrenedivinyl) benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABXTM resin (J. T. Baker, Phillipsburg, N.J. ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-
exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column) , chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
Following any preliminary purification step (s) , the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt) .
Purification
In certain embodiments, the antibodies of the present disclosure may be purified. The term “purified, ” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it may naturally occur. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%or more of the proteins in the composition.
Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) . Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. Other methods for protein purification include, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; gel filtration, reverse phase, hydroxylapatite and affinity chromatography; and combinations of such and other techniques.
In purifying an antibody of the present disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column, which binds to a tagged portion of the polypeptide. As is generally known in the art, it is believed that the order of conducting the various purification steps may be
changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
Commonly, complete antibodies are fractionated utilizing agents (i.e., protein A) that bind the Fc portion of the antibody. Alternatively, antigens may be used to simultaneously purify and select appropriate antibodies. Such methods often utilize the selection agent bound to a support, such as a column, filter or bead. The antibodies are bound to a support, contaminants removed (e.g., washed away) , and the antibodies released by applying conditions (salt, heat, etc. ) .
Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity. The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977) . It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
III. Pharmaceutical Composition
The present disclosure further provides pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof described herein and one or more pharmaceutically acceptable carriers.
Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxanisol, butylated hydroxytoluene, and/or propyl gallate. As disclosed herein, inclusion of one or more antioxidants such as methionine in a composition comprising an antibody or antigen-binding fragment and conjugates as provided herein decreases oxidation of the antibody or antigen-binding fragment. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf-life. Therefore, in certain embodiments compositions are provided that comprise one or more antibodies or antigen-binding fragments as disclosed herein and one or more antioxidants such as methionine. Further provided are methods for preventing oxidation of, extending the shelf-life of, and/or improving the efficacy of an antibody or antigen-binding fragment as provided herein by mixing the antibody or antigen-binding fragment with one or more antioxidants such as methionine.
To further illustrate, pharmaceutical acceptable carriers may include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid) , ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
The pharmaceutical compositions can be a liquid solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
In certain embodiments, the pharmaceutical compositions are formulated into an injectable composition. The injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion. Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions. The solutions may be either aqueous or nonaqueous.
In certain embodiments, unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides a desirable formulation. In one embodiment, the resulting solution will be apportioned into vials for lyophilization. Each vial can contain a single dosage or multiple dosages of the bispecific antibody or antigen-binding fragment thereof or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ℃ to room temperature.
Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration. In one embodiment, for reconstitution the
sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
In certain embodiments, the pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof described herein further comprise one or more additional therapeutic agents that are co-administered with the bispecific antibodies or antigen-binding fragments thereof. The candidates of the additional therapeutic agents are disclosed infra in Section IV. It can be understood that the additional therapeutic agents can be co-formulated with the bispecific antibodies or antigen-binding fragments thereof, or be mixed with the bispecific antibodies or antigen-binding fragments thereof right before the administration, such as in the IV infusion bag.
IV. Methods of Use of Bispecific Antibodies
The present disclosure also provides therapeutic methods comprising: administering a therapeutically effective amount of the antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing cancer.
Examples of cancer can be generally categorized into solid tumors and hematologic malignancies. Solid tumors include but are not limited to, non-small cell lung cancer (squamous/non-squamous) , small cell lung cancer, renal cell cancer, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma) , pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, multiple myeloma, mycoses fungoides, Merkel cell cancer, hepatocellular carcinoma (HCC) , fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma/synovial sarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, mast cell derived tumors, EBV-positive and -negative PTLD, nasopharyngeal carcinoma, spinal axis tumor, brain stem glioma, astrocytoma,
medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.
Solid tumors are characterized by multiple biologic hallmarks including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, activating invasion and metastasis, tumor promoting inflammation, avoiding immune destruction, genomic instability and mutation, and deregulating cellular energetics. Treatment efforts have evolved from cytotoxic chemotherapies targeting rapidly dividing cells to small molecules inhibiting select signaling pathways to monoclonal antibodies targeting surface proteins. More recently the concept of cancer immunotherapy to reinvigorate endogenous immunity or cellular therapies utilizing synthetic immunity have shown promise. Despite these advances, most patients with advanced solid tumors still do not survive long-term. The use of immune checkpoint inhibitors such as anti-CTLA-4 or anti-PD-1/PD-L1 have led to long-term progression-free and overall survival in a minority of patients.
Hematologic malignancies include but are not limited to acute lymphocytic/lymphoblastic leukemia (ALL) , acute myeloid leukemia (AML) , B-cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN) , chronic lymphoblastic leukemia (CLL) , chronic lymphocytic leukemia (CLL) , chronic myeloid leukemia (CML) , chronic myelomonocytic leukemia (CMML) , classical Hodgkin lymphoma (CHL) , diffuse large B-cell lymphoma (DLBCL) , extranodal NK/T-cell lymphoma, hairy cell leukemia, heavy chain disease, HHV8-associated primary effusion lymphoma, lymphoid malignancy, multiple myeloma (MM) , myelodysplasia, myelodysplastic syndrome (MDS) , non-Hodgkin's lymphoma, plasmablastic lymphoma, pre-B acute lymphocytic leukemia (Pre-B ALL) , primary CNS lymphoma, primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, myeloproliferative neoplasms, and Waldenstrom's macroglobulinemia.
The therapeutically effective amount of an antibody or antigen-binding fragment as provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
In certain embodiments, the antibody or antigen-binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.0001 mg/kg to about 100 mg/kg. In certain of these embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less. In certain embodiments, the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) . For example, a single dose may be administered, or several divided doses may be administered over time.
The antibodies and antigen-binding fragments disclosed herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
In some embodiments, the antibodies or antigen-binding fragments disclosed herein may be administered alone or in combination with one or more additional therapeutic means or agents. For example, the antibodies or antigen-binding fragments disclosed herein may be administered in combination with another therapeutic agent, for example, a chemotherapeutic agent or an anti-cancer drug.
In certain of these embodiments, an antibody or antigen-binding fragment as disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments the antibody or antigen-binding fragment and the additional therapeutic agent (s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment administered “in combination” with another therapeutic agent does not have to be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the antibody or antigen-binding fragment and second agent are administered via different routes. Where possible, additional therapeutic agents administered
in combination with the antibodies or antigen-binding fragments disclosed herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Prescriber’s Digital Reference (available online only at pdr. net) or protocols well known in the art.
In certain embodiments, the agent for combination therapy is an anti-neoplastic composition. As used herein, an “anti-neoplastic composition” refers to a composition useful in treating cancer comprising at least one active therapeutic agent. Examples of therapeutic agents include, but are not limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, cancer immunotherapeutic agents, apoptotic agents, anti-tubulin agents, and other-agents to treat cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor) , HER1/EGFR inhibitor (e.g., erlotinibplatelet derived growth factor inhibitors (e.g., (Imatinib Mesylate) ) , a COX-2 inhibitor (e.g., celecoxib) , interferons, CTLA4 inhibitors (e.g., anti-CTLA antibody ipilimumabor tremelimumab) , PD-l or PD-L1 inhibitors (e.g., or nivolumab, or pembrolizumab, or atezolizumab, or avelumab, or durvalumab, or cemiplimab-rwlc, or sintilimab, tislelizumab (BGB-A317) , penpulimab (AK105) , camrelizumab, toripalimab, zimberelimab (GLS-010) , retifanlimab, sugemalimab, or CS1003) , dual-targeting antibodies against CTLA-4 and PD-1 or PD-L1 (e.g., an anti-PD-1/CTLA-4 bi-specific antibody or AK104) , TIM3 inhibitors (e.g., anti-TIM3 antibodies) , LAG-3 inhibitors (e.g., anti-LAG3 antibodies) , cytokines, TLR agonists, STING agonists, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, FGFR2b, PDGFR-beta, BlyS, APRIL, BCMA, or VEGF receptor (s) , TRAIL/Apo2, an IDH1 inhibitor, an ivosidenib, an IDH2 inhibitor, an enasidenib, asmoothened (SMO) inhibitor, a glasdegib, an arginase inhibitor, an IDO inhibitor, an epacadostat, a BCL-2 inihbitor, a venetoclax, aplatinum complex derivative, oxaliplatin, a kinase inhibitor, a tyrosine kinase inhibitor, a PI3 kinase inhibitor, a BTK inhibitor, an ibrutinib, an acalabrutinib, azanubrutinib, an ICOS antibody, a TIGIT antibody, a CD40 antibody, a 4-1BB antibody, a Siglec antibody, an OX40 antibody, a TNFR2 antibody, an antibody to another LILR family member, a CD47 antibody, a SIRP1α antibody or fusions protein, an antagonist of E-selectin, an antibody binding to a tumor antigen, an antibody binding to a T cell surface marker, an antibody binding to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an
alkaloid derived from a plant, a hormone therapy medicine, a hormone antagonist, an aromatase inhibitor, a P-glycoprotein inhibitor and other bioactive and organic chemical agents, etc., an engineered T cell, NK cell or macrophage, a bispecific antibody.
In certain embodiments, the agent for combination therapy is a chemotherapeutic agent. As used herein, a “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents that can be administered in methods herein include, but are not limited to, alkylating agents such as thiotepa andcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone) ; a camptothecin (including the synthetic analogue topotecan) ; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues) ; cryptophycins (particularly cryptophycin 1 and cryptophycin 8) ; dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1) ; eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl., 33: 183-186 (1994) ) ; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores) , aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin) , epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti metabolites such as methotrexate and 5-fluorouracil (5-FU) ; folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex (JHS Natural Products, Eugene, OR) ; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ( “Ara-C” ) ; cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J. ) , Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois) , anddoxetaxel (Rhone-Poulenc Rorer, Antony, France) ; chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16) ; ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin) ; topoisomerase inhibitor RFS 2000; difluorometlhylomithine (DMFO) ; retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV) ; oxabplatin, including the oxaliplatin treatment regimen (FOLFOX) ; inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Further nonlimiting exemplary chemotherapeutic agents that can be administered in methods herein include anti-hormonal agents that act to regulate or inhibit hormone action on cancers such as anti-estrogens and selective estrogen receptor modulators (SERMs) , including, for example, tamoxifen (includingtamoxifen) , raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie,
fadrozole, vorozole, letrozole, andanastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a l, 3-dioxolane nucleoside cytosine analog) ; antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes such as a VEGF expression inhibitor (e.g., ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapy vaccines, for example, vaccine, vaccine, andvaccine; rIL-2; topoisomerase 1 inhibitor; rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
In certain embodiments, the agent for combination therapy is an anti-angiogenesis agent. As used herein, an “anti-angiogenesis agent” refers to a small molecular weight substance, a polynucleotide (including, e.g., an inhibitory RNA (RNAi or siRNA) ) , a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly. It should be understood that the anti-angiogenesis agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor. For example, an anti-angiogenesis agent that can be administered in methods herein can include an antibody or other antagonist to an angiogenic agent, e.g., antibodies to VEGF-A (e.g., bevacizumab) or to the VEGF-A receptor (e.g., KDR receptor or Flt-l receptor) , anti-PDGFR inhibitors such as (Imatinib Mesylate) , small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, /SUl 1248 (sunitinib malate) , AMG706, or those described in, e.g., international patent application WO 2004/113304) . Anti-angiogenesis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D’A more (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene 22: 3172-3179; Ferrara &Alitalo (1999) Nature Medicine 5 (12) : 1359-1364; Tonini et al. (2003) Oncogene 22: 6549-6556; and Sato (2003) Int. J.Clin. Oncol. 8: 200-206.
In certain embodiments, the agent for combination therapy is a growth inhibitory agent. As used herein, a “growth inhibitory agent” as used herein refers to a compound or composition that inhibits growth of a cell (such as a cell expressing VEGF) either in vitro or in vivo. Thus, the growth inhibitory agent that can be administered in methods herein may be one that significantly reduces the percentage of cells (such as a cell expressing VEGF) in S phase. Examples of growth inhibitory agents include, but are not limited to, agents that
block cell cycle progression (at a place other than S phase) , such as agents that induce Gl arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine) , taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (W. B. Saunders, Philadelphia, 1995) , e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (Rhone-Poulenc Rorer) , derived from the European yew, is a semisynthetic analogue of paclitaxel (Bristol-Myers Squibb) . Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
The dose of the agent for the combination therapy can be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular agent. Typically, the attending physician will decide the dosage of the agent for the combination therapy with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, the agent be administered, route of administration, and the severity of the condition being treated. By way of example and not intending to limit the present disclosure, the dose for the combination therapy can be about 0.0001 to about 1 g/kg body weight of the subject being treated/day, from about 0.0001 to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg bodyweight/day. Dosage units may be also expressed in mg/m2, which refer to the quantity in milligrams per square meter of body surface area.
Each therapeutic agent in the combination therapy described herein may be administered simultaneously (e.g., in the same medicament or at the same time) , concurrently (i.e., in separate medicaments administered one right after the other in any order or sequentially in any order. Sequential administration may be useful when the therapeutic agents in the combination therapy are in different dosage forms (one agent is a tablet or capsule and another agent is a sterile liquid) and/or are administered on different dosing schedules, e.g., a chemotherapeutic that is administered at least daily and a biotherapeutic that is administered less frequently, such as once weekly, once every two weeks, or once every three weeks.
In certain embodiments, the bispecific antibody of the present disclosure and the second drug are combined or co-formulated in a single dosage form. In certain embodiments, the bispecific antibody of the present disclosure and the second drug are administered separately. Although the simultaneous administration of the bispecific antibody of the present disclosure and the second drug may be maintained throughout a period of treatment, anti-cancer activity may also be achieved by subsequent administration of one compound in isolation (for example, the bispecific antibody following initial combination treatment, or alternatively, the second drug following initial combination treatment) . In some embodiments, the bispecific antibody is administered before administration of the second drug, while in other embodiments, the bispecific antibody is administered after administration of the second drug. In some embodiments, at least one of the therapeutic agents in the combination therapy is administered using the same dosage regimen (dose, frequency and duration of treatment) that is typically employed when the agent is used as monotherapy for treating the same cancer. In other embodiments, the patient receives a lower total amount of at least one of the therapeutic agents in the combination therapy than when the agent is used as monotherapy, e.g., smaller doses, less frequent doses, and/or shorter treatment duration.
The combination therapy of the invention may be used prior to or following surgery to remove a tumor and may be used prior to, during or after radiation therapy. The combination therapy of the invention may be used to treat a tumor that is large enough to be found by palpation or by imaging techniques well known in the art, such as MRI, ultrasound, or CAT scan. In some embodiments, the combination therapy of the invention is used to treat an advanced stage tumor having dimensions of at least about 200 mm3, 300 mm3, 400 mm3, 500 mm3, 750 mm3, or up to 1000 mm3.
In some embodiments, the present disclosure also provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating cancer in a subject.
EXAMPLES
While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments) , it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.
Example 1: Construction and expression of anti-CD4 and anti-PD-L1 bispecific antibodies
The sequence of anti-CD4 antibody is from patent US Patent No. 11,254,745 B1, disclosure of which is incorporated herein by reference in their entirety. The sequence of anti-PD-L1 antibody is from patent US Patent No. 8,217,149 B2, disclosure of which is incorporated herein by reference in their entirety. Anti-CD4 and anti-PD-L1 bispecific antibodies were constructed by fusing the single chain variable fragment (scFv) of anti-PD-L1 antibody (atezolizumab) to the carboxyl-terminus of heavy chain (2B6-hIgG1/Atezo-scFv) (FIG. 1) or light chain (2B6-hkappa/Atezo-scFv) (FIG. 2) of anti-CD4 antibody (2B6) . The heavy chain and light chain sequence of 2B6-hIgG1/Atezo-scFv bispecific antibody are shown in SEQ ID NO: 21 and SEQ ID NO: 10, respectively. The heavy chain and light chain sequence of 2B6-hkappa/Atezo-scFv bispecific antibody are shown in SEQ ID NO:9 and SEQ ID NO: 22, respectively. DNAs encoding 2B6-hIgG1/Atezo-scFv and 2B6-hkappa/Atezo-scFv antibody light chain and heavy chain were synthesized and cloned to the expression vector pcDNA3.1 (Invitrogen, Cat No: V-790) . Freestyle 293 cells (1000 mL at 106/mL) were transfected with 1000 μg of each of the heavy and light chain expression plasmids and cultured for 6 days at 37℃. The bispecific antibody in the supernatant was then purified with Protein-A column (GE healthcare) .
2B6-hIgG1 (SEQ ID NO: 9)
Atezo-scFv (SEQ ID NO: 20)
2B6-hIgG1/Atezo-scFv (SEQ ID NO: 21) (linkers are underlined below) :
2B6-hkappa (SEQ ID NO: 10)
2B6-hkappa/Atezo-scFv (SEQ ID NO: 22) (linkers are underlined below) :
Example 2: ELISA based binding analysis of anti-CD4/anti-PD-L1 bispecific antibody
ELISA binding analysis was conducted by using human CD4-mFc or PD-L1-mFc as antigen. 96-well plates (Costar, Cat No: 9018) were coated with 100 μL of 2 μg/ml CD4-mFc or PD-L1-mFc in PBS coating buffer (Hyclone, Cat No: SH30256.01B) overnight at 4℃.The wells were aspirated and non-specific binding sites were blocked by adding 200 μL of blocking buffer (PBS with 1% (w/v) of bovine serum albumin (BSA, Roche, Cat No: 738328) ) and incubating for 1 hour at 37℃. After the plates were washed three times with wash buffer (PBS with 0.05% (v/v) Tween20 (Sigma, Cat No: P1379) ) , 100 μL/well of 1: 10 serial dilutions
of anti-CD4/anti-PD-L1 bispecific antibody in blocking buffer (starting from 20 μg/mL) was added and incubated at room temperature for 1 hour. The plates were washed and incubated with 100 μL/well of Goat anti-Mouse IgG (H+L) (Thermo, Cat No: 31432) in blocking buffer for 60 min. After the plates were washed, 100 μL/well of substrate solution TMB (eBioscience, Cat No: 00-4201-56) were added and the plates were incubated for 2min at room temperature. 100 μL/well of stop solution (2N H2SO4) were added to stop the reaction. The colorimetric signals were developed and read at 450 nm using an Auto Plate SpectraMax Plus (Supplier: Moleculer Devices; Model: MNR0643; Software: SoftMax Pro v5.4) , and the data was analyzed using GraphPad Prism 5. As shown in FIGs. 3-4 and Table 3-4, 2B6-hIgG1/Atezo-scFv and 2B6-hkappa/Atezo-scFv bispecific antibody can bind to both CD4 and PD-L1 with an EC50 comparable to the corresponding monoclonal antibody.
Table 3. Binding EC50 of anti-CD4/anti-PD-L1 bispecific antibody with CD4
Table 4. Binding EC50 of anti-CD4/anti-PD-L1 bispecific antibody with PD-L1
Example 3: Binding kinetic study of anti-CD4/anti-PD-L1 bispecific antibody with CD4 and PD-L1
The binding kinetics of bispecific antibody with CD4 and PD-L1 were measured by surface plasmon resonance (SPR) analysis, which was performed at 25℃ on a Biacore T200 instrument. Protein A (GE, Cat. No: 29139131-AA) was diluted with 10 mM pH 5.0 sodium acetate and immobilized onto reference and experiment flow cells of a CM5 biosensor chip to around 15000RU using an amine coupling kit (GE, BR10050) . In the beginning of each cycle, diluted bispecific antibody (1.5 μg/mL) was injected over experiment flow cell for 1 minute to be captured. CD4-HisTag and PD-L1-HisTag analyte series were prepared by diluting the stocks with running buffer to 100 nM followed by 2-fold serial dilution in the same buffer down to 0.78 nM. Analytes were injected in series over the reference and experiment flow cells
for 3 minutes at a flow rate of 30 μL/minute. Running buffer (PBS with 0.05%P20) was allowed to flow over for 10 minutes at a flow rate of 30 μL/minute. At the end of each cycle, the biosensor surface was regenerated with 3-minute injection of 10 mM pH 2.0 Glycine-HCl buffer at a flow rate of 10 μL/minute. For each analyte sample injection (i.e. each cycle) , binding responses obtained from the experimental biosensor surface were double referenced by subtracting simultaneously recorded responses from the reference surface followed by additional subtraction of responses from a single referenced running buffer sample. The association and dissociation rate constants (ka and kd) were determined simultaneously by fitting double-referenced sensorgrams of the entire titration series to Langmuir model (1: 1) using Biaevaluation software. The dissociation constant, KD, was calculated from the determined rate constants by the relation KD = kd/ka. The binding affinity of anti-CD4/anti-PD-L1 bispecific antibody with human CD4-HisTag and PD-L1-HisTag are summarized in Table 5-6.
Table 5 Binding kinetics of anti-CD4/anti-PD-L1 bispecific antibody with CD4
Table 6 Binding kinetics of anti-CD4/anti-PD-L1 bispecific antibody with PD-L1
Example 4: Effect of anti-CD4/anti-PD-L1 bispecific antibody on T cell activation in the mixed lymphocyte reaction
Human CD4+ T-cells were purified from human PBMC using a CD4+ negative selection isolation kit (Mitenyi Biotech, Cat No: 130-091-155) . Immature dendritic cells (DC) were derived from monocytes isolated from human PBMC using the Mo-DC Generation Toolbox (Miltenyi,
Cat No: 130-093-568) . The cells were cultured with Mo-DC Differentiation Medium for 7 days, and were then induced to be mature DC with Mo-Dc Maturation medium for 2 days. For mixed lymphocyte reaction setting-up, each reaction was added with 105 purified T-cells and 104 allogeneic mature DC cells in a total volume of 200 μL. The bispecific antibodies were assayed at different concentrations (0.02, 0.2, 2, 20 μg/mL) , and an IgG1 isotype control antibody was used as a negative control. The cells were cultured for 5 days at 37 ℃. On day 3, the levels of IFN-γ and IL-2 in the culture medium were measured using the IL-2 ELISA kit (eBioscience) and hIFN-γ ELISA kit (R&D, Cat No: DY285) . The results are shown in FIG. 5 for IL-2 secretion, and FIG. 6 for IFN-γ secretion. Both 2B6-hIgG1/Atezo-scFv and 2B6-hkappa/Atezo-scFv bispecific antibody promoted T-cell IFN-gamma and IL-2 secretion in a concentration dependent manner.
Example 5: Antibody-dependent cell-mediated cytotoxicity effect of anti-CD4/anti-PD-L1 bispecific antibody
The ability of anti-CD4/anti-PD-L1 bispecific antibody to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was assessed based on the killing of CD4 positive cells in PBMC. In 96-well plate, PBMCs (2×10^5 cells/well) were incubated with series concentrations of anti-CD4/anti-PD-L1 bispecific 2 antibody (160 ng/mL, 16 ng/mL, 5.333 ng/mL, 1.778ng/mL, 0.178 ng/mL, 0.018 ng/mL) at 37℃, 5%CO2 for 24 hours. AF-488 labeled CD4 antibody (OKT4-AF488, the binding epitope of OKT4 antibody does not overlap with the CD4 antibody 2B6) was then added to each well and incubated at 4 ℃ for 1 hour, and living CD4+ cells in each well were detected with FACS. The ADCC effect was shown in FIG. 7 by calculating with the following formula:
ADCC%= (1-No. of living CD4 positive cells in the presence of antibody/No. of living CD4 positive cells in the absence of antibody) ×100%
As shown in FIG. 7 and Table 7, 2B6-hIgG1/Atezo-scFv and 2B6-hkappa/Atezo-scFv bispecific antibody can induce ADCC with an EC50 comparable to the corresponding monoclonal antibody 2B6.
Table 7 The ADCC activity of anti-CD4/anti-PD-L1 bispecific antibody
*************
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Claims (33)
- An antibody or an antigen-binding fragment thereof, comprising(a) a CD4 binding domain comprising(i) a heavy chain (HC) variable region (VH) comprising a HCDR1 comprising the sequence set forth in SEQ ID NO: 1, a HCDR2 comprising the sequence set forth in SEQ ID NO: 2, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 3, and(ii) a light chain (LC) variable region (VL) comprising a LCDR1 comprising the sequence set forth in SEQ ID NO: 4, a LCDR2 comprising the sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising the sequence set forth in SEQ ID NO: 6; and(b) a PD-L1 binding domain comprising(i) a VH comprising a HCDR1 comprising the sequence set forth in SEQ ID NO: 11, a HCDR2 comprising the sequence set forth in SEQ ID NO: 12, and a HCDR3 comprising the sequence set forth in SEQ ID NO: 13, and(ii) a VL comprising a LCDR1 comprising the sequence set forth in SEQ ID NO: 14, a LCDR2 comprising the sequence set forth in SEQ ID NO: 15, and a LCDR3 comprising the sequence set forth in SEQ ID NO: 16.
- The antibody or an antigen-binding fragment thereof of claim 1, wherein the VH of the CD4 binding domain comprises the sequence set forth in SEQ ID NO: 7 and the VL of the CD4 binding domain comprises the sequence set forth in SEQ ID NO: 8.
- The antibody or an antigen-binding fragment thereof of claim 1, wherein the VH of the PD-L1 binding domain comprises the sequence set forth in SEQ ID NO: 17 and VL of the PD-L1 binding domain comprises the sequence set forth in SEQ ID NO: 18.
- The antibody or an antigen-binding fragment thereof of claim 1, wherein the VH of the CD4 binding domain is linked to a heavy chain constant region and the VL of the CD4 binding domain is linked to a light chain constant region.
- The antibody or an antigen-binding fragment thereof of claim 4, wherein the heavy chain constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4.
- The antibody or an antigen-binding fragment thereof of claim 4, wherein the heavy chain constant region comprises a constant region of human IgG1.
- The antibody or an antigen-binding fragment thereof of claim 6, wherein the IgG1 comprises one or more mutations that can confer increased CDC or ADCC relative to wild-type constant region.
- The antibody or an antigen-binding fragment thereof of claim 7, wherein the one or more mutations is selected from the group consisting of S239D, I332E, H268F, S324T S236A, G236A, P247I, A339 (D/Q) , D280H, K290S, S298 (D/V) , F243L, R292P, Y300L, P396L, V305I, K290 (E/N) , S298G, T299A, K326E, E382V, M428I, S298A, K326A, E333A, and K334A, according to EU numbering.
- The antibody or antigen-binding fragment thereof of claim 8, wherein the one or more mutations comprise a combination of S298A, E333A, and K334A, according to EU numbering.
- The antibody or antigen-binding fragment thereof of claim 8, wherein the one or more mutations comprise a combination of S239D, A330L, and I332E, according to EU numbering.
- The antibody or an antigen-binding fragment thereof of claim 1, wherein the PD-L1 binding domain is a scFv (single chain fragment variable) domain.
- The antibody or an antigen-binding fragment thereof of claim 11, wherein the scFv domain is linked to a heavy chain constant region.
- The antibody or an antigen-binding fragment thereof of claim 11, wherein the scFv domain is linked to a light chain constant region.
- The antibody or an antigen-binding fragment thereof of claim 1, comprising(a) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 21; and(b) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 10.
- The antibody or an antigen-binding fragment thereof of claim 1, comprising(a) a heavy chain polypeptide comprising the sequence set forth in SEQ ID NO: 9; and(b) a light chain polypeptide comprising the sequence set forth in SEQ ID NO: 22.
- The antibody or an antigen-binding fragment thereof of claim 1, wherein the antibody is a murine, a rodent, a rabbit, a chimeric, a humanized, or a human antibody.
- The antibody or an antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment comprises a recombinant scFv domain, a Fab fragment, a F (ab’) 2 fragment, or a Fv fragment.
- The antibody or an antigen-binding fragment thereof of any of the preceding claims, which is humanized.
- The antibody or an antigen-binding fragment thereof of any of the preceding claims, which is linked to one or more conjugate moieties.
- The antibody or an antigen-binding fragment thereof of claim 19, wherein the conjugate moiety comprises an immune modulatory agent, an anti-tumor drug, a clearance-modifying agent, a toxin, a detectable label, an RNA, a DNA, a cytokine, or purification moiety.
- A pharmaceutical composition comprising the isolated antibody or an antigen-binding fragment thereof of any of the preceding claims, and a pharmaceutically acceptable carrier.
- An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of claims 1-20.
- A vector comprising the isolated polynucleotide of claim 22.
- A host cell comprising the vector of claim 23.
- A method of producing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 24 under the condition at which the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.
- A method of treating or ameliorating the effect of a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any of claims 1-20, or the pharmaceutical composition of claim 20.
- The method of claim 26, wherein the cancer is selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer.
- The method of claim 26 or 27, wherein the subject is human.
- The method of claim 26 or 27, wherein the antibody or an antigen-binding fragment thereof is administered intravenously, intra-arterially, intra-tumorally, intra-muscularly, or subcutaneously.
- The method of claim 26 or 27, further comprising administering to the subject one or more drugs selected from the group consisting of a chemotherapeutic agent, a growth inhibitory agent, a cytotoxic agent, an agent used in radiation therapy, an anti-angiogenesis agent, a cancer immunotherapeutic agent, a apoptotic agent, an anti-tubulin agent, an anti-HER-2 antibody, an anti-CD20 antibody, an epidermal growth factor receptor (EGFR) antagonist, HER1/EGFR inhibitor, a platelet derived growth factor inhibitor, a COX-2 inhibitor, an interferon, a CTLA4 inhibitor (e.g., anti-CTLA antibody ipilimumabor tremelimumab) , a PD-l or PD-L1 inhibitor (e.g., or nivolumab, or pembrolizumab, or atezolizumab, or avelumab, or durvalumab, or cemiplimab-rwlc, or sintilimab, tislelizumab (BGB-A317) , penpulimab (AK105) , camrelizumab, toripalimab, zimberelimab (GLS-010) , retifanlimab, sugemalimab, or CS1003) , a dual-targeting antibody against CTLA-4 and PD-1 or PD-L1 (e.g., an anti-PD-1/CTLA-4 bi-specific antibody or AK104) , a TIM3 inhibitor (e.g., anti-TIM3 antibodies) , a LAG-3 inhibitor (e.g., anti-LAG3 antibodies) , a cytokine, an antagonist (e.g., neutralizing antibodies) that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, FGFR2b, PDGFR-beta, BlyS, APRIL, BCMA, or VEGF receptor (s) , TRAIL/Apo2, an IDH1 inhibitor, an ivosidenib, an IDH2 inhibitor, an enasidenib, asmoothened (SMO) inhibitor, a glasdegib, an arginase inhibitor, an IDO inhibitor, an epacadostat, a BCL-2 inihbitor, a venetoclax, aplatinum complex derivative, oxaliplatin, a kinase inhibitor, a tyrosine kinase inhibitor, a PI3 kinase inhibitor, a BTK inhibitor, an ibrutinib, an acalabrutinib, azanubrutinib, a TLR agonist, a STING agonist, an ICOS antibody, a TIGIT antibody, a CD40 antibody, a 4-1BB antibody, a CD47 antibody, an OX40 antibody, a TNFR2 antibody, an antibody to another LILR family member, a Siglec antibody, a SIRP1α antibody or fusions protein, an antagonist of E-selectin, an antibody binding to a tumor antigen, an antibody binding to a T cell surface marker, an antibody binding to a myeloid cell or NK cell surface marker, an alkylating agent, a nitrosourea agent, an antimetabolite, an antitumor antibiotic, an alkaloid derived from a plant, a hormone therapy medicine, a hormone antagonist, an aromatase inhibitor, and a P-glycoprotein inhibitor, an engineered T cell, NK cell or macrophage.
- The method of claim 30, wherein the one or more drugs are co-administered with the antibody or an antigen-binding fragment thereof.
- The method of claim 31, wherein the one or more drugs are (a) co-formulated with the antibody or an antigen-binding fragment thereof or (b) pre-mixed with the antibody or an antigen-binding fragment thereof in intravenous (IV) infusion bag, prior to administering to the subject.
- Use of the antibody or antigen-binding fragment thereof of any of claims 1-20 in the manufacture of a medication for treating cancer in a subject.
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