CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the priority of Chinese Patent Application No. 202110373640.7, filed Apr. 7, 2021, the disclosure of which is incorporated herein by reference in the entirety.

SEQUENCE LISTING

The sequence listing that is contained in the file named “056211-8004US01_SL_ST25”, which is 38 KB (as measured in Microsoft Windows) and was created on May 25, 2021, is filed herewith by electronic submission and is incorporated by reference herein.

BACKGROUND

1. Field of the Invention

The present disclosure generally relates to antibodies and antigen-binding fragments thereof that bind to human CD4, and to methods of using such antibodies and antigen-binding fragments.

2. Description of Related Art

CD4 is a monomeric type I transmembrane glycoprotein and a member of the immunoglobulin supergene family, and it comprises 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 C E, et al., Proc Natl Acad Sci USA (1988) 85: 5190; Barber E K, et al., Proc Natl Acad Sci USA (1989) 86: 3277 and Li Q J, 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 M N, 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). Therefore, there is a need for novel anti-CD4 antibodies that can effectively target and eliminate the CD4-expressing cells, especially Tregs.

BRIEF SUMMARY OF THE INVENTION

Throughout the present disclosure, the articles “a,” “an,” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an antibody” means one antibody or more than one antibody.

The present disclosure provides, among others, an isolated antibody against human CD4 (hCD4) or an antigen-binding fragment thereof, comprising a heavy chain variable (VH) region and/or a light chain variable (VL) region, wherein the heavy chain variable region comprises:a) a HCDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 11, 21, 31 and 41,b) a HCDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 12, 22 and 42, andc) a HCDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 3, 13, 23, 33 and 43, and/or
wherein the light chain variable region comprises:d) a LCDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 14, 24 and 34,e) a LCDR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 5, 15 and 25, andf) a LCDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 6, 16, 26 and 46.

In certain embodiments, the heavy chain variable region is selected from the group consisting of:a) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 2, and a HCDR3 comprising the sequence of SEQ ID NO: 3;b) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 11, a HCDR2 comprising the sequence of SEQ ID NO: 12, and a HCDR3 comprising the sequence of SEQ ID NO: 13;c) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 21, a HCDR2 comprising the sequence of SEQ ID NO: 22, and a HCDR3 comprising the sequence of SEQ ID NO: 23;d) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 31, a HCDR2 comprising the sequence of SEQ ID NO: 2, and a HCDR3 comprising the sequence of SEQ ID NO: 33; ande) a heavy chain variable region comprising a HCDR1 comprising the sequence of SEQ ID NO: 41, a HCDR2 comprising the sequence of SEQ ID NO: 42, and a HCDR3 comprising the sequence of SEQ ID NO: 43.

In certain embodiments, the light chain variable region is selected from the group consisting of:a) a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 4, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;b) a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 14, a LCDR2 comprising the sequence of SEQ ID NO: 15, and a LCDR3 comprising the sequence of SEQ ID NO: 16;c) a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 24, a LCDR2 comprising the sequence of SEQ ID NO: 25, and a LCDR3 comprising the sequence of SEQ ID NO: 26;d) a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 34, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6; ande) a light chain variable region comprising a LCDR1 comprising the sequence of SEQ ID NO: 24, a LCDR2 comprising the sequence of SEQ ID NO: 25, and a LCDR3 comprising the sequence of SEQ ID NO: 46.

In certain embodiments, the antibody or an antigen-binding fragment thereof provided herein comprises:a) a heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO: 1, a HCDR2 comprising the sequence of SEQ ID NO: 2, and a HCDR3 comprising the sequence of SEQ ID NO: 3; and a light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 4, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6;b) a heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO: 11, a HCDR2 comprising the sequence of SEQ ID NO: 12, and a HCDR3 comprising the sequence of SEQ ID NO: 13; and a light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 14, a LCDR2 comprising the sequence of SEQ ID NO: 15, and a LCDR3 comprising the sequence of SEQ ID NO: 16;c) a heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO: 21, a HCDR2 comprising the sequence of SEQ ID NO: 22, and a HCDR3 comprising the sequence of SEQ ID NO: 23; and a light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 24, a LCDR2 comprising the sequence of SEQ ID NO: 25, and a LCDR3 comprising the sequence of SEQ ID NO: 26;d) a heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO: 31, a HCDR2 comprising the sequence of SEQ ID NO: 2, and a HCDR3 comprising the sequence of SEQ ID NO: 33; and a light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 34, a LCDR2 comprising the sequence of SEQ ID NO: 5, and a LCDR3 comprising the sequence of SEQ ID NO: 6; ore) a heavy chain variable region comprises a HCDR1 comprising the sequence of SEQ ID NO: 41, a HCDR2 comprising the sequence of SEQ ID NO: 42, and a HCDR3 comprising the sequence of SEQ ID NO: 43; and a light chain variable region comprises a LCDR1 comprising the sequence of SEQ ID NO: 24, a LCDR2 comprising the sequence of SEQ ID NO: 25, and a LCDR3 comprising the sequence of SEQ ID NO: 46.

In certain embodiments, the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 61, and SEQ ID NO: 63, and a homologous sequence thereof having at least 80% sequence identity yet retaining specific binding specificity or affinity to CD4.

In certain embodiments, the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 62, and SEQ ID NO: 64, and a homologous sequence thereof having at least 80% sequence identity yet retaining specific binding specificity or affinity to CD4.

In certain embodiments, the antibody or an antigen-binding fragment thereof provided herein comprises:a) a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and a light chain variable region comprising the sequence of SEQ ID NO: 8; orb) a heavy chain variable region comprising a sequence of SEQ ID NO: 17 and a light chain variable region comprising a sequence of SEQ ID NO: 18; orc) a heavy chain variable region comprising a sequence of SEQ ID NO: 27 and a light chain variable region comprising a sequence of SEQ ID NO: 28; ord) a heavy chain variable region comprising a sequence of SEQ ID NO: 37 and a light chain variable region comprising a sequence of SEQ ID NO: 38; ore) a heavy chain variable region comprising a sequence of SEQ ID NO: 47 and a light chain variable region comprising a sequence of SEQ ID NO: 48; orf) a heavy chain variable region comprising a sequence of SEQ ID NO: 61 and a light chain variable region comprising a sequence of SEQ ID NO: 62; org) a heavy chain variable region comprising a sequence of SEQ ID NO: 63 and a light chain variable region comprising a sequence of SEQ ID NO: 64.

In another aspect, the present disclosure provides an isolated antibody against human CD4 (hCD4) or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising the sequence of SEQ ID NO: 65 and a light chain variable region comprising the sequence of SEQ ID NO: 66, and a homologous sequence thereof having at least 80% sequence identity yet retaining specific binding specificity or affinity to CD4.

In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises one or more amino acid residue substitutions or modifications yet retains specific binding specificity or affinity to hCD4.

In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences, and/or in one or more of the non-CDR regions of the VH or VL sequences.

In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises an immunoglobulin constant region, optionally a constant region of human Ig, or optionally a constant region of human IgG.

In certain embodiments, the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4.

In certain embodiments, the 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 certain embodiments, the antibody or an antigen-binding fragment thereof provided herein is humanized.

In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is a diabody, a Fab, a Fab′, a F(ab′)2, a Fd, 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 multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody.

In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is bispecific.

In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is capable of specifically binding to a first and a second epitope of hCD4, or capable of specifically binding to both hCD4 and a second antigen.

In certain embodiments, the second antigen comprises an immune related target.

In certain embodiments, the second antigen comprises PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, A2AR, CD160, 2B4, TGF β, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CD28, CD30, CD40, CD47, CD122, ICAM-1, IDO, NKG2C, SLAMF7, SIGLEC7, NKp80, CD160, B7-H3, LFA-1, 1COS, 4-1BB, GITR, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-7, IL-15, IL-21, CD3, CD16 or CD83.

In certain embodiments, the second antigen comprises a tumor antigen.

In certain embodiments, the tumor antigen comprises a tumor specific antigen or a tumor associated antigen.

In certain embodiments, the tumor antigen comprises prostate specific antigen (PSA), CA-125, gangliosides G(D2), G(M2) and G(D3), CD20, CD52, CD33, Ep-CAM, CEA, bombesin-like peptides, HER2/neu, epidermal growth factor receptor (EGFR), erbB2, erbB3/HER3, erbB4, CD44v6, Ki-67, cancer-associated mucin, VEGF, VEGFRs (e.g., VEGFR-1, VEGFR-2, VEGFR-3), estrogen receptors, Lewis-Y antigen, TGF-beta1, IGF-1 receptor, EGF, c-Kit receptor, transferrin receptor, Claudin 18.2, GPC-3, Nectin-4, ROR1, methothelin, PCMA, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, BCR-ABL, E2APRL, H4-RET, IGH-IGK, MYL-RAR, IL-2R, CO17-1A, TROP2, or LIV-1.

In certain embodiments, the antibody or antigen-binding fragment thereof provided herein is linked to one or more conjugate moieties.

In certain embodiments, the conjugate moiety comprises a clearance-modifying agent, a chemotherapeutic agent, a toxin, a radioactive isotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme-substrate label, a DNA-alkylator, a topoisomerase inhibitor, a tubulin-binders, or other anticancer drugs such as androgen receptor inhibitor.

In another aspect, the present disclosure provides an antibody or an antigen-binding fragment thereof, which competes for binding to hCD4 with the antibody or antigen-binding fragment thereof provided herein.

In another aspect, the present disclosure provides a pharmaceutical composition or kit comprising the antibody or antigen-binding fragment thereof provided herein, and a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition or kit provided herein further comprises a second therapeutic agent.

In certain embodiments, the second therapeutic agent comprises an anti-cancer agent, such as a mTOR inhibitor, an immune checkpoint inhibitor and a T cell-recruiting antibody.

In certain embodiments, the mTOR inhibitor is Temsirolimus, Evirolimus or Rapamycin.

In certain embodiments, the immune checkpoint inhibitor is selected from an PD-1 antibody, PD-L1 antibody, PD-L2 antibody, LAG-3 antibody, TIM-1 antibody, CTLA-4 antibody, VISTA antibody, B7-H2 antibody, B7-H3 antibody, B7-H4 antibody, B7-H antibody 6, ICOS antibody, HVEM antibody, CD160 antibody, gp49B antibody, PIR-B antibody, KIR family receptors antibody, TIM-1 antibody, TIM-4 antibody, BTLA antibody, SIRPalpha (CD47) antibody, CD244 antibody, B7.1 antibody, B7.2 antibody, ILT-2 antibody, ILT-4 antibody, TIGIT antibody and A2aR antibody.

In certain embodiments, the T cell-recruiting antibody comprises a CD19/CD3 bispecific antibody.

In another aspect, the present disclosure provides an isolated polynucleotide encoding the antibody or an antigen-binding fragment thereof provided herein.

In another aspect, the present disclosure provides a vector comprising the isolated polynucleotide provided herein.

In another aspect, the present disclosure provides a host cell comprising the vector provided herein.

In another aspect, the present disclosure provides a method of expressing the antibody or antigen-binding fragment thereof provided herein, comprising culturing the host cell under the condition at which the vector is expressed.

In another aspect, the present disclosure provides a method of treating a CD4-related disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the pharmaceutical composition provided herein.

In certain embodiments, the CD4-related disease or condition is cancer, adaptive immune disease, autoimmune disease, inflammatory disease, or infectious disease.

In certain embodiments, the cancer is selected from the group consisting of lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicle cancer, kidney cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervix cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, stomach cancer, vagina cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, adenocarcinoma, leukemia (e.g., chronic lymphocytic leukemia, relapsed or refractory B-cell precursor acute lymphoblastic leukemia), myeloma and lymphoma.

In another aspect, the present disclosure provides a method of detecting presence or amount of CD4 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof provided herein, and determining the presence or the amount of CD4 in the sample.

In another aspect, the present disclosure provides a method of diagnosing a CD4-related disease or condition in a subject, comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with the antibody or antigen-binding fragment thereof provided herein; c) determining presence or amount of CD4 in the sample; and d) correlating the presence or the amount of CD4 to existence or status of the CD4 related disease or condition in the subject.

In another aspect, the present disclosure provides a method of eliminating CD4-expressing cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the pharmaceutical composition provided herein.

In certain embodiments, the CD4-expressing cell is regulatory T cell (Tregs).

In another aspect, the present disclosure provides a method of enhancing immunogenicity of tumor microenvironment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the pharmaceutical composition provided herein.

In another aspect, the present disclosure provides a method of improving the therapeutic efficacy of immunotherapy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof or the pharmaceutical composition provided herein.

In certain embodiments, the method provided herein further comprises administering a second therapeutic agent.

In certain embodiments, the second therapeutic agent comprises an anti-cancer agent, such as a m TOR inhibitor, an immune checkpoint inhibitor and a T cell-recruiting antibody.

In certain embodiments, the T cell-recruiting antibody comprises a CD19/CD3 bispecific antibody.

In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD4-related disease or condition in a subject.

In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for enhancing immunogenicity of tumor microenvironment in a subject in need thereof.

In another aspect, the present disclosure provides use of the antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for improving the therapeutic efficacy of immunotherapy in a subject in need thereof.

