Activatable bispecific anti-CD3 and anti-PD-L1 proteins and uses thereof

Abstract

Provided herein is a protein molecule that specifically binds PD-L1 and exhibits activatable specific CD3 binding in diseased tissue.Further provided herein is the use of such protein molecule to treat cancer.Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, and a second CH1 domain; the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL domain, and a second immunoglobulin light chain constant region.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 325,437, filed March 20, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (ULSL_004_01WO_SeqList_ST26.xml; size: 173,185 bytes; and creation date: March 29, 2023) are incorporated by reference herein in their entirety.

[0003] Technical Field The present disclosure relates to activatable bispecific proteins and treatments for cancer. [Background technology]

[0004] background In immune oncology therapy, most of the important drug targets are not exclusively expressed in diseased tissues, and the major ones are also expressed in non-diseased tissues. Moreover, many drugs used in cancer treatment use very powerful cell killing mechanisms. As a result, the binding of targets by drugs in non-diseased tissues often causes undesirable side effects.

[0005] PD-L1 is a cell surface receptor that is a member of the immunoglobulin superfamily and is expressed primarily on myeloid cells and regulatory T (Treg) cells in non-diseased tissues. However, PD-L1 has also been observed to be highly expressed on some cancer cells. PD-L1 binds to the membrane protein PD1. The interaction of PD-L1 with PD1 on T cells downregulates T cell inflammatory activity, which promotes immune self-tolerance. PD-L1 is therefore described as an immune checkpoint. Thus, antagonistic anti-PD-L1 monoclonal antibodies that block the interaction with PD1 have demonstrated the potential to act as well-tolerated immunotherapeutic agents in disease settings such as cancer by "liberating" anti-cancer T cell responses from the restrictions of innate immunity. Thus, PD-L1 is a drug target used to amplify the anti-cancer effects of the adaptive immune system. However, in many cases, tumors can use multiple "escape mechanisms" to negate the effects of PD-L1 drugs, such as downregulation of MHC class 1 and downregulation of cancer neoantigen expression.

[0006] CD3 (cluster of differentiation 3) is a T cell coreceptor involved in activating both CD8+ and CD4+ T cells as part of the T cell receptor complex. It is composed of four separate chains, including two CD3ε chains. Historically, antibodies have been generated against the CD3ε chain, which upon binding can induce TCR activation signals in T lymphocytes that can result in increased cytolytic activity against infected or cancerous cells. Using binding domains derived from these antibodies, CD3-linked bispecific agents have also been generated that can direct the killing activity of T cells to target-specific cell classes without the need for TCR recognition of MHC-presented antigens on target cells. Thus, anti-PD-L1 antibodies could become more potent and broadly acting therapeutic agents by acquiring the ability to also bind CD3ε, thereby strongly engaging T cell killing mechanisms in PD-L1 antibody-resistant disease settings. This combines an important checkpoint inhibitor function with an inducible "synthetic immunity" that can synergistically stimulate the adaptive immune system. However, the ability to make this combination work in a single therapeutic construct (e.g., in a standard bispecific antibody format with fully active PD-L1 and CD3 binding domains) is limited by the relatively broad expression profile of both PD-L1 and CD3 on many cell types (e.g., T cells and myeloid cells, etc.). This broad expression profile not only results in dose-limiting toxicity of PD-L1 / CD3 binding agents, but can also pose significant peripheral sink / biodistribution problems that limit the ability of such agents to achieve high enough exposure in diseased tissues to take advantage of their combined mechanisms. Thus, there is a need for engineered forms of bispecific binding proteins that have activity that is specifically targeted to the diseased tissue environment. Summary of the Invention [Means for solving the problem]

[0007] (Summary) Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) Domain, a first CH1 Domain, a first linker, an anti-CD3 VH Domain, and a second CH1 Domain; and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL Domain, and a second immunoglobulin light chain constant region.

[0008] Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) Domain, a first CH1 Domain, a first linker, an anti-CD3 light chain variable (VL) Domain, and a first immunoglobulin light chain constant region, and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a second immunoglobulin light chain constant region, a second linker, an anti-CD3 VH Domain, and a second CH1 Domain.

[0009] In some embodiments, the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0010] In some embodiments, the protein further comprises a third polypeptide chain comprising a hinge, a CH2 domain, and a CH3 domain. In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124.

[0011] The proteins provided herein further comprise a moiety that provides half-life extension. In some embodiments, the moiety that provides half-life extension is polyethylene glycol (PEG) or an albumin binding domain.

[0012] Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) Domain, a first CH1 Domain, a first linker, an anti-CD3 VH Domain, a second CH1 Domain, a first hinge, a first CH2 Domain, and a first CH3 Domain; and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL Domain, a second immunoglobulin light chain constant region, a second hinge, a second CH2 Domain, and a second CH3 Domain.

[0013] In some embodiments, the first linker comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 12. In some embodiments, the second linker comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 12.

[0014] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0015] In some embodiments, the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0016] In some embodiments, the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0017] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:20, an HCDR2 comprising the amino acid sequence of SEQ ID NO:21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:23, an LCDR2 comprising the amino acid sequence of SEQ ID NO:24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:25; the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; and the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0018] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:20, an HCDR2 comprising the amino acid sequence of SEQ ID NO:21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:23, an LCDR2 comprising the amino acid sequence of SEQ ID NO:24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:25; the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; and the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0019] In some embodiments, the anti-PD-L1 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 33 and 54 to 107. In some embodiments, the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 34.

[0020] In some embodiments, the anti-PD-L1 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 33 and 54-107, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 34.

[0021] In some embodiments, the anti-CD3 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 44 to 48. In some embodiments, the anti-CD3 VL domain comprises the amino acid sequence of any one of SEQ ID NOs: 37 to 42.

[0022] In some embodiments, the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 116-123, 129-132, 137-141, 142, 144, 146, 147, 148, 150, and 152. In some embodiments, the light chain comprises the amino acid sequence of any one of SEQ ID NOs: 53, 108-115, 125-128, 133-136, 143, 145, 151, and 153.

[0023] (a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 122, the light chain comprises the amino acid sequence of SEQ ID NO: 114, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (b) the heavy chain comprises the amino acid sequence of SEQ ID NO: 123, the light chain comprises the amino acid sequence of SEQ ID NO: 115, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (c) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (d) the heavy chain comprises the amino acid sequence of SEQ ID NO: 117, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (e) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (f) the heavy chain comprises the amino acid sequence of SEQ ID NO: 119, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (g) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (h) the heavy chain comprises the amino acid sequence of SEQ ID NO:116, the light chain comprises the amino acid sequence of SEQ ID NO:110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:124; (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (j) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (k) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (l) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (m) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (n) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 112, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (o) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 113, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (p) the heavy chain comprises the amino acid sequence of SEQ ID NO: 121, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (q) the heavy chain comprises the amino acid sequence of SEQ ID NO: 131, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; or (r) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; Proteins are provided herein.

[0024] In some embodiments, the heavy chain comprises an IgG, IgE, IgM, IgD, IgA, or IgY constant region. In some embodiments, the heavy chain comprises an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 constant region. In some embodiments, the heavy chain comprises an immunologically inert constant region. In some embodiments, the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region with amino acid substitutions L234A, L235A, and G237A, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, or a human IgG4 constant region with amino acid substitution S228P, where numbering is according to the EU index as in Kabat.

[0025] Provided herein are immunoconjugates comprising a protein disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent.

[0026] Provided herein are pharmaceutical compositions comprising a protein, or immunoconjugate, disclosed herein, and a pharma- ceutically acceptable carrier.

[0027] Provided herein are nucleic acid molecules that encode: (a) the first polypeptide chain amino acid sequence; (b) the second polypeptide chain amino acid sequence; or (c) the amino acid sequences of both the first and second polypeptide chains of the proteins disclosed herein.

[0028] Provided herein are nucleic acid molecules encoding (a) the heavy chain amino acid sequence; (b) the light chain amino acid sequence; or (c) both the heavy and light chain amino acid sequences of the proteins disclosed herein.

[0029] Provided herein are expression vectors comprising the nucleic acid molecules disclosed herein.

[0030] Provided herein are recombinant host cells comprising the nucleic acid molecules or expression vectors disclosed herein.

[0031] Provided herein are methods of producing a protein, the method comprising culturing a recombinant host cell disclosed herein under conditions in which a nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0032] Provided herein is a method for enhancing an anti-cancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0033] Provided herein are methods for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0034] Provided herein is a method for ameliorating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0035] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in enhancing an anti-cancer immune response in a subject.

[0036] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in treating cancer in a subject.

[0037] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in ameliorating a symptom of cancer in a subject.

[0038] In some embodiments of the methods and uses provided herein, the methods provided herein are wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue. [Brief description of the drawings]

[0039] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A]FIG. 1A shows schematic diagrams of protein molecules disclosed herein in intact (left), activating protease-cleaved (middle), and non-activating protease-cleaved (right) conformations. In the intact conformation, the PD-L1 Fab binding domain is exposed and can bind their cognate target. The CD3 Fab domain is inhibited from binding by linkers in both the heavy and light chains, both of which are proteolytically cleavable and can be sequentially cleaved by matrix metalloproteases (MMPs) and / or cathepsins. The first cleavage event creates an intermediate active state that allows both PD-L1 Fab and CD3 Fab derived from a single protein construct to bind their cognate targets, thereby potentially directing the killing of PD-L1+ cells by directed activation of CD3+ T cells. The second cleavage releases the PD-L1 Fab and CD3 Fab, eliminating the ability of a single molecule to bind both PD-L1 and CD3 simultaneously.

[0040] [Figure 1B] FIG. 1B shows a detailed diagram of an intact asymmetric LB (lock body) protein consisting of a heavy chain, a light chain and an Fc base. Each light chain is composed of two Fabs linked by a low hinge linker (LHL). Each heavy chain is composed of two Fabs linked by a low hinge linker (LHL), an Fc hinge and an Fc fragment containing both CH2 and CH3 domains with either an N-linked glycosylation site (CH2) or a knob mutation (CH3). Finally, the asymmetric LB protein contains a third polypeptide containing both CH2 and CH3 domains with either an N-linked glycosylation site (CH2) or a Holes mutation (CH3).

[0041] [Figure 1C]FIG. 1C shows a schematic diagram of an alternative format of the protein molecule disclosed herein, where the molecule may lack an Fc fragment (left); may lack an Fc sequence but achieve half-life extension (HLE) through alternative mechanisms such as PEGylation, addition of an albumin binding domain, etc. (middle); or may contain an Fc fragment but be constructed from only two polypeptides as a symmetric one-arm construct (right). Each polypeptide chain comprises either two light chain Fabs linked by a low hinge linker, a disulfide-bond-free Fc hinge and an Fc fragment (CH2 and CH3 domains), or two heavy chain Fabs linked by a low hinge linker, a disulfide-bond-free Fc hinge and an Fc fragment (CH2 and CH3 domains).

[0042] [Figure 1D] Figure ID shows a detailed diagram of an asymmetric one-arm construct in which two polypeptide chains comprise one light and one heavy Fab linked by an LHL. As above, each polypeptide chain further comprises a disulfide-free Fc hinge and an Fc fragment (CH2 and CH3 domains).

[0043] [Diagram 2] FIG. 2 shows a schematic diagram of one proposed mechanism of activity of the activatable bispecific protein molecules provided herein.

[0044] [Figure 3A]Figures 3A-G show the activity of (1) an IgG1 isotype, (2) a commercially available CD3xHer2 bispecific T cell engager (BiTE) used as a positive control anti-Her2 / anti-CD3 bispecific antibody as indicated, or (3) a test article (before treatment with MMP12 or at 0, 0.5, 1 or 4 hours after treatment) in a cell-based assay measuring the ability of agents to bind hPD-L1 on MDA-MB231 cancer cells and induce CD3 signaling in co-cultured Jurkat cells (measured by fold activation). Figure 3A shows the activity of an IgG1 isotype, a CD3xHer2 BiTE used as a positive control, and the test article LB204 (before treatment with MMP12 or at 0 or 4 hours after treatment). LB204 induces low CD3 signal before MMP12 treatment and moderate CD3 activation fold after MMP12 treatment, which remains below the level induced by commercial CD3xHer2 BiTE control. Figure 3B shows the activity of IgG1 isotype, CD3xHer2 BiTE used as positive control and test article LB206 (before treatment with MMP12, or after 0 hours or 4 hours of treatment). LB206 induces low CD3 signal before MMP12 treatment and induces high CD3 signal after treatment with MMP12, which is higher than the level of commercial CD3xHer2 BiTE control. Figure 3C shows the activity of IgG1 isotype, CD3xHer2 BiTE used as positive control and test article LB208 (before treatment with MMP12, or after 0 hours or 4 hours of treatment). LB208 induces no CD3 signal before MMP12 treatment and induces a moderate CD3 activation fold after MMP12 treatment that remains below the level induced by the commercial CD3xHer2 BiTE control. Figure 3D shows the activity of the IgG1 isotype, the CD3xHer2 BiTE used as a positive control, and the test article LB209 (before treatment with MMP12 or after 0 or 4 hours of treatment).LB209 does not induce CD3 signal before MMP12 treatment, and induces a moderate CD3 activation fold after MMP12 treatment, which remains below the level induced by the commercial CD3xHer2 BiTE control. Figure 3E shows the activity of IgG1 isotype, CD3xHer2 BiTE used as positive control, and test article LB210 (before treatment with MMP12, or after 0 hours or 4 hours of treatment). LB210 does not induce CD3 signal before or after MMP12 treatment. Figure 3F shows the activity of IgG1 isotype, CD3xHer2 BiTE used as positive control, and test article LB213 (before treatment with MMP12, or after 0 hours or 4 hours of treatment). LB213 does not induce CD3 signal before MMP12 treatment, and induces a relatively high CD3 signal after MMP12 treatment, but remains below the level induced by the commercial CD3xHer2 BiTE control. Figure 3G shows the activity of the IgG1 isotype, test article LB205 (before treatment with MMP12 or after 0, 0.5 or 1 hour treatment). LB205 induces no CD3 signal before MMP12 treatment and induces high CD3 signal after either 0.5 or 1 hour treatment with MMP12. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above.

[0045] [Figure 4A]Figure 4A-D show the characterization of the Fab and IgG formats of the two CD3 humanized variants (SPR, ELISA, and Jurkat activity). Figure 4A shows the surface plasmon resonance (SPR) binding of each V domain humanized variant (in monovalent Fab format) to human recombinant CD3 δε heterodimers. Figure 4B shows the surface plasmon resonance (SPR) binding of each V domain humanized variant (in monovalent Fab format) to cynomolgus monkey (cyno) recombinant CD3 δε heterodimers. Figure 4C shows the IgG binding of IgG001 and IgG002 to human and cyno recombinant CD3 δε heterodimers in ELISA. Figure 4D shows the CD3-dependent activation assay comparing the luciferase signal emitted from Jurkat reporter cells stimulated with SP34 IgG or its respective humanized variant in IgG format. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above.

[0046] [Figure 5A] Figure 5A shows MMP12 digested LB proteins run on SDS PAGE under reducing or non-reducing conditions. 2 μg / lane of exemplary LB proteins 204, 206, 208, 209, 210 and 213 pre-incubated with MMP12 for 0 and 4 hours shows differential digestion profiles. Notably, prominent 25 kDa / 50 kDa bands in reducing / non-reducing SDS-PAGE, respectively, arise from light chain cleavage, and the intact heavy chain at 75 kDa in reducing conditions (and 150 kDa in non-reducing conditions) disappears, which can be seen in concert, indicating that the linkers in both the heavy and light chains are cleaved.

[0047] [Figure 5B]FIG. 5B shows MMP12 digested LB proteins run on SDS PAGE under reducing or non-reducing conditions. 2 μg / lane of LB proteins, LB206 and LB220, pre-incubated with MMP12 for 0 h, 5 min, 15 min, 30 min, 60 min or 120 min show differential digestion profiles. Notably, prominent 25 kDa / 50 kDa bands in reducing / non-reducing SDS-PAGE, respectively, result from light chain cleavage, and disappearance of intact heavy chain at 75 kDa in reducing conditions (and 150 kDa in non-reducing conditions), which can be observed in concert, indicating that linkers in both heavy and light chains are cleaved. In direct comparison, LB protein LB220 appears to be cleaved by MMP12 at a faster rate than LB206.

[0048] [Figure 6A]Figures 6A-C show the activity of IgG1 isotype or test article (before or after 0, 0.5 or 1 hour treatment with MMP12) in a cell-based assay measuring the ability of test agents to bind hPD-L1 on MDA-MB231 cancer cells and induce CD3 signaling in co-cultured Jurkat cells (measured by fold activation). Figure 6A shows the activity of IgG1 isotype, test article LB217 (before or after 0, 0.5 or 1 hour treatment with MMP12). LB217 induces a low CD3 signal before MMP12 treatment and a high CD3 signal after either 0.5 or 1 hour treatment with MMP12. Figure 6B shows the activity of IgG1 isotype, test article LB218 (before or after 0, 0.5 or 1 hour treatment with MMP12). LB217 does not induce CD3 signal before MMP12 treatment, and induces high CD3 signal after 0.5 hours of MMP12 treatment. In contrast, CD3 signal after 1 hour of MMP12 treatment is low. Figure 6C shows the activity of IgG1 isotype, test article LB220 (before treatment with MMP12, or after 0 hours, 0.5 hours or 1 hour of treatment). LB220 induces low CD3 signal before MMP12 treatment, and induces high CD3 signal after 0.5 hours of MMP12 treatment. In contrast, CD3 signal after 1 hour of MMP12 treatment is much lower, similar to that without MMP12 treatment. [Figure 6B] Same as above. [Figure 6C] Same as above.

[0049] [Figure 7A]Figures 7A-H show ELISA binding of LB proteins, either intact or incubated with MMP12 for up to 1 hour, to human PD-L1, with atezolizumab as a positive control, or human and cyno CD3 δε heterodimers. The negative control protein used is human IgG1 isotype. Figure 7A shows ELISA binding of LB206 proteins, either intact or incubated with MMP12 for 5 minutes, to human PD-L1, with atezolizumab as a positive control, or IgG1 isotype (no signal) as a negative control. Intact and MMP12-treated LB206 bind to PD-L1 to similar levels. Figure 7B shows ELISA binding of LB206 proteins, either intact or incubated with MMP12 for 5 minutes, to human CD3 δε heterodimers, or IgG1 isotype (no signal) as a negative control. Intact LB206 binds to human CD3 δε heterodimers at very low levels. In contrast, MMP12-treated LB206 binds strongly to human CD3 δε heterodimer. Figure 7C shows ELISA binding of LB218 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 minutes, to human PD-L1, using IgG1 isotype (no signal) as a negative control. Intact and MMP12-treated LB218 for 5 or 15 minutes bind to PD-L1 at similar levels. PD-L1 appears to be low at 30 and 60 minutes. Figure 7D shows ELISA binding of LB218 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 minutes, to human CD3 δε heterodimer, using IgG1 isotype (no signal) as a negative control. Intact LB218 shows very low levels of binding to human CD3 δε heterodimer. As the MMP12 treatment time is increased, higher binding to human CD3 δε heterodimers can be observed, with LB218 treated with MMP12 for 60 minutes showing the highest binding to human CD3 δε heterodimers.Figure 7E shows ELISA binding of LB218 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 minutes, to cyno CD3 δε heterodimer, using IgG1 isotype (no signal) as a negative control. Intact LB218 shows very low levels of binding to cyno CD3 δε heterodimer. Higher binding to cyno CD3 δε heterodimer can be observed with increasing MMP12 treatment time. LB218 treated with MMP12 for 60 minutes shows the highest binding to cyno CD3 δε heterodimer. Figure 7F shows ELISA binding of LB213 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 minutes, using IgG1 isotype (no signal) as a negative control, to human PD-L1. Intact and LB213 treated with MMP12 for 5 or 15 minutes bind to PD-L1 at similar levels. Binding to PD-L1 appears to be low at 30 and 60 min, suggesting the occurrence of proteolytic cleavage 2 (Figure 1A). Figure 7G shows ELISA binding of LB213 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 min, to human CD3 δε heterodimer, with IgG1 isotype (no signal) as a negative control. Intact LB213 shows binding to human CD3 δε heterodimer to very low levels. Higher binding to human CD3 δε heterodimer can be observed with increasing MMP12 treatment time. LB213 treated with MMP12 for 60 min shows higher binding to human CD3 δε heterodimer. Figure 7H shows ELISA binding of LB213 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 min, to cyno CD3 δε heterodimers, using the IgG1 isotype (no signal) as a negative control. Intact LB213 shows very low levels of binding to cyno CD3 δε heterodimers.As the MMP12 treatment time is increased, higher binding to the cyno CD3 δε heterodimer can be observed. LB213 cells treated with MMP12 for 60 minutes show higher binding to the cyno CD3 δε heterodimer. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] Same as above. [Figure 7H] Same as above.

[0050] [Figure 8A] Figure 8A shows Jurkat cell binding of the LB protein LB206, either intact or incubated with MMP12 for 0-2 hours. No binding to Jurkat cells can be observed for intact LB206 (0 min) or the isotype control. In contrast, all MMP12-treated samples show similar binding levels to Jurkat cells. Figure 8B shows PDL-1 expression on three different cell lines - A549, MDA-MB-231 and RKO cells after IFNγ stimulation. [Figure 8B] Same as above.

[0051] [Figure 9A]9A-9F show primary T cell killing of cancer cell lines (A549, RKO, MDA-MB-231) mediated by LB proteins with the indicated concentrations either intact or incubated with MMP12 as measured by the Incucyte® live cell analysis platform. FIG. 9A shows primary T cell killing of cancer cell line A549 mediated by LB 206 either intact or incubated with MMP12 for 30 minutes as measured by the Incucyte® live cell analysis platform. FIG. 9B shows primary T cell killing of cancer cell line A549 mediated by LB218 either intact or incubated with MMP12 for 30 minutes as measured by the Incucyte® live cell analysis platform. FIG. 9C shows primary T cell killing of the cancer cell line RKO mediated by LB206 either intact or incubated with MMP12 for 30 minutes as measured by the Incucyte® live cell analysis platform. FIG. 9D shows primary T cell killing of the cancer cell line RKO mediated by LB218 either intact or incubated with MMP12 for 30 minutes as measured by the Incucyte® live cell analysis platform. FIG. 9E shows primary T cell killing of the cancer cell line MDA-MB-231 mediated by LB206 either intact or incubated with MMP12 for 30 minutes as measured by the Incucyte® live cell analysis platform. FIG. 9F shows primary T cell killing of the cancer cell line MDA-MB-231 mediated by LB213 either intact or incubated with MMP12 for 30 minutes as measured by the Incucyte® live cell analysis platform. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above.

[0052] [Figure 10A]Figures 10A-10E show the change in tumor volume (baseline corrected) or body weight over time in CD34+ myeloid boosted NCG mice bearing established MDA-MB-231 tumors while treated with LB206, LB213, LB220, atezolizumab or IgG isotype control. Figure 10A shows the tumor volume over time in CD34+ myeloid boosted NCG mice bearing established MDA-MB-231 tumors treated with LB206, IgG isotype control or atezolizumab. All LB206 treatment groups (4.5 mg / kg, 8.5 mg / kg, 12 mg / kg) show tumor regression to a similar extent. FIG. 10B shows the caliper-measured tumor volume over time of CD34+ myeloid-boosted NCG mice with established MDA-MB-231 tumors treated with LB220, IgG isotype control, or atezolizumab. All LB220-treated groups (8.5 mg / kg, 12 mg / kg) show initial tumor regression followed by tumor growth inhibition. FIG. 10C shows the caliper-measured tumor volume over time of CD34+ myeloid-boosted NCG mice with established MDA-MB-231 tumors treated with LB213, IgG isotype control, or atezolizumab. All LB213-treated groups show dose-dependent tumor growth inhibition (4.5 mg / kg, 8.5 mg / kg, 12 mg / kg). FIG 10D shows tumor volumes of individual tumors in CD34+ myeloid boosted NCG mice treated with either IgG isotype or LB206 through day 51. Dosing regimens are indicated by triangles. Five of eight LB206 treated tumors continue to regress 33 days after treatment was stopped. FIG 10E shows the average body weight change over time for all treatment groups. None of the groups showed more than 10% weight loss at any time. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Detailed Description With respect to oncology, there are two key issues that limit the efficacy of CD3-activating, tumor-targeted bispecific antibody drugs:

[0054] 1) Antibody targeting proteins found on cancer cells (e.g., PD-L1) are potentially expressed on many different cell classes in the body, not just tumor cells. This off-tumor targeting expression often poses the risk of dose-limiting side effects, because the CD3 binding domain in standard bispecific molecules is constitutively active and thus can instruct T cells to kill any target-positive cell, whether it is in diseased tissue or not. Off-tumor targeting expression can also result in an antigen "sink" effect, which reduces the amount of drug penetrating the tumor.

[0055] 2) CD3 positive cells are found in high concentrations in the bloodstream and in secondary lymphoid tissues, causing a large sink effect for this arm and affecting free drug availability for biodistribution and tumor penetration.

[0056] Both factors described above minimize the potential stability and efficacy of bispecific antibodies driving CD3 engagement and activation. The anti-PD-L1 and anti-CD3 proteins provided herein (see, e.g., Figures 1A-1D) overcome the peripheral sink and toxicity issues by minimizing CD3 binding except in diseased tissues. This effect is achieved by adding a PD-L1 binding domain and linker above (i.e., n-terminal) the CD3 binding domain. Use of the appropriate upper domain and linker combination results in a configuration that minimizes binding activity at the lower (i.e., c-terminal) CD3 domain. The PD-L1 domain then drives enrichment in the PD-L1-enriched tumor microenvironment. The protein construct linker system exploits the elevated MMP and cathepsin activity common in solid tumors to cleave the linker peptide and expose the CD3 binding domain, thereby conditionally activating CD3 activation activity in the tumor but not in the periphery. These combined biological functions thereby endow the molecule with the potential to circumvent peripheral CD3 sinks and maximize T cell immune responses against cancer cells, as outlined in FIG. 2.

[0057] Provided herein is a protein that is conditionally active in diseased human tissue. The protein of the present disclosure is fully active in specifically binding and blocking PD-L1 throughout the body; shows minimized binding of CD3 in healthy tissue; and is highly activated in binding and activation of CD3 once in the PD-L1 positive diseased tissue environment. The protein of the present disclosure comprises a CD3 binding domain that is masked by the PD-L1 binding domain in non-diseased tissue. The protein also comprises two peptide linkers that are cleaved by one or more proteases expressed in diseased tissue (e.g., tumor). The linker cleavage unmasks the CD3 binding domain in diseased tissue, thus selectively enabling the protein to bind and / or function in the diseased tissue.

[0058] Protein molecules Provided herein is a protein that comprises two Fab fragments, an anti-PD-L1 Fab and an anti-CD3 Fab, which is monovalent when in the intact structure and can only have at most monovalent CD3 binding when activated, to minimize the risk of peripheral toxicity associated with bivalent activating anti-CD3 antibodies.

[0059] In some embodiments, the protein comprises a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, the heavy chain comprising, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, and a second CH1 domain; the light chain comprising, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL domain, and a second immunoglobulin light chain constant region. In some embodiments, the heavy chain further comprises an immunoglobulin hinge region and an Fc domain at its C-terminus. In some embodiments, the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0060] In some embodiments, the protein further comprises a third polypeptide chain comprising a hinge, a CH2 domain, and a CH3 domain. The third polypeptide chain may be referred to as an "Fc-stump."

[0061] A schematic of an exemplary protein of the disclosure is shown in FIG. 1 with the domains indicated. FIG. 1A shows schematics of the protein molecules disclosed herein in intact (left), activating protease-cleaved (middle) and non-activating protease-cleaved (right) conformations. In the intact conformation, the PD-L1 Fab binding domain is exposed and can bind their cognate target. The CD3 Fab domain is inhibited from binding by linkers in both the heavy and light chains, both of which are proteolytically cleavable and can be sequentially cleaved by matrix metalloproteases (MMPs) and / or cathepsins. The first cleavage event creates an intermediate active state that allows both PD-L1 Fab and CD3 Fab derived from a single protein construct to bind their cognate targets, thereby potentially directing the killing of PD-L1+ cells by directed activation of CD3+ T cells. The second cleavage dissociates the PD-L1 Fab and CD3 Fab, eliminating the ability of a single molecule to bind both PD-L1 and CD3 simultaneously. Figure 1B shows a detailed view of an intact asymmetric LB (lock body) protein consisting of a heavy chain, a light chain and an Fc base. Each light chain is composed of two Fabs linked by a low hinge linker (LHL). Each heavy chain is composed of two Fabs linked by a low hinge linker (LHL), an Fc hinge and an Fc fragment containing both CH2 and CH3 domains with either an N-linked glycosylation site (CH2) or a knob mutation (CH3). Finally, the asymmetric LB protein contains a third polypeptide containing both CH2 and CH3 domains with either an N-linked glycosylation site (CH2) or a hole mutation (CH3). Figure 1C shows a schematic diagram of an alternative format of the protein molecule disclosed herein. Here the molecule may lack an Fc fragment (left); may lack an Fc fragment but achieve half-life extension (HLE) through alternative mechanisms such as PEGylation, addition of an albumin binding domain, etc. (center); may include an Fc fragment but be constructed from only two polypeptides as a symmetric one-arm construct (right).Each polypeptide chain comprises either two light chain Fabs linked by a low hinge linker, a disulfide-bond free Fc hinge and an Fc fragment (CH2 and CH3 domains), or two heavy chain Fabs linked by a low hinge linker, a disulfide-bond free Fc hinge and an Fc fragment (CH2 and CH3 domains). Figure ID shows a detailed diagram of an asymmetric one-arm construct in which the two polypeptide chains comprise one light chain and one heavy chain Fab linked by an LHL. As above, each polypeptide chain further comprises a disulfide-bond free Fc hinge and an Fc fragment (CH2 and CH3 domains).

[0062] The first linker and the second linker are cleavable by matrix metalloproteases (MMPs) and / or cathepsins found in diseased tissues (e.g., tumors). The linkers in the protein are immunoglobulin-derived hinge sequences that are proteolytically sensitive and can be sequentially cleaved; the first cleavage utilizes an intact structure and creates an intermediate active state that allows the anti-PD-L1 Fab and anti-CD3 Fab from a single protein construct to bind their cognate targets. The second cleavage event in the second linker removes the covalent bond between the anti-PD-L1 Fab and the anti-CD3 Fab, revoking the ability of the molecule to recruit T cell killing of PD-L1+ cells. This second cleavage event constitutes a "self-destruct mechanism" that thereby minimizes the risk of the activated molecule escaping the tumor microenvironment. Cleaved linkers based on immunoglobulin hinge sequences can also recruit increased immune effector functions (antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP)) at the cell membrane via endogenous anti-hinge antibodies, a known phenomenon in human patients with (and even without) underlying autoreactive disease.

[0063] In some embodiments, the protein comprises one or more amino acid sequences provided in Table 1, Table 2, Table 6, Table 7, or Table 8. [Table 1-1] [Table 1-2] [Table 1-3] In the VH and VL domain sequences, the CDR sequences are underlined. [Table 2-1] [Table 2-2] Bold / underlined = humanized residues. The 6L and 5H sequences are provided in WO 2014 / 167022. LC = light chain HC=heavy chain

[0064] Table 3 describes selected Fab proteins generated with selected sequences from Table 2 as described. Both anti-CD3 Fab proteins were further characterized in SPR binding experiments as described in FIG. 4A. [Table 3]

[0065] Table 4 describes selected IgG proteins generated with selected sequences from Table 2 as described. Both CD3 IgG proteins were further characterized in ELISA binding experiments and Jurkat reporter assays as described in Figures 4B and 4C. [Table 4]

[0066] Table 5 lists the anti-PD-L1 VH variants. Individual point mutations in the CDRs were generated by in silico modeling and are summarized in Table 5. The purpose of these anti-PD-L1 variants was affinity engineered for cynomolgus monkey and / or human PD-L1. [Table 5] X1=T, R, Q X2=A, S, H, L X3=G, N, H X4 = I, F X5=I, K, W, L, Y, Q, R X6= G, H, Q, N X7=K, I, F, M, D, Y, L, W, H, R X8=H, L X9=Q, E X 10 =R, K, F, N, H, P, Q X 11 =S, W, R, L, Q, G, Y, D, N, A, M X 12 =G, A, V, S, Q, N X 13 =S,P

[0067] Table 6 shows the selected anti-PD-L1 VH variant heavy domains based on Table 5. These anti-PD-L1 variants were affinity matured to cynomolgus and / or human PD-L1. Expression of each of the VH variable domains together with the wild-type (WT) VL variable domain in Fab format was performed followed by determination of the KD to human and cynomolgus PD-L1 in SPR. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0068] Table 7 shows an exemplary full length LB protein, light chain and heavy chain sequences. Each chain comprises a WT anti-PD-L1 variable domain followed by an LHL and CD3 Fab variant. For the asymmetric one-arm construct, the heavy chain, Fc hinge and associated Fc fragment are present as depicted in Figure 1A and Figure 1B (right). For the construct depicted in Figure 1B (left, center), each chain comprises a WT anti-PD-L1 variable domain followed by an LHL and CD3 Fab variant. For the symmetric one-arm construct, both the light and heavy chain sequences are followed by an Fc hinge and Fc fragment as depicted in Figure 1C. Each polypeptide chain in Table 7 is named with: first, a number from 1 to 7 (Table 2) referring to the respective CD3 humanized chain used; second, by either HH or LL, or B-HH or B-LL referring to asymmetric or symmetric constructs (B-); and third, a letter of choice from X, F, RF or RX-F (which indicates details regarding the LHL sequence, as specified in Table 1). Finally, the addition of HLE in this table refers to the option of adding the appropriate half-life extending moiety. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9]

[0069] Table 8 shows an exemplary full-length LB protein sequence as shown in Figure ID. Each chain comprises, from N- to C-terminus, a WT anti-PD-L1 variable domain, followed by an LHL and CD3 Fab variant, an Fc linker and an Fc fragment. In contrast to the sequences listed in Table 7, each chain comprises heavy and light chain variable domains as outlined and illustrated in Figure ID. [Table 8-1] [Table 8-2] [Table 8-3]

[0070] The above anti-PD-L1 / anti-CD3 protein designs may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and may or may not have effector function capabilities.

[0071] In some embodiments, the proteins disclosed herein comprise an Fc fragment. In some embodiments, the proteins disclosed herein do not comprise an Fc fragment.

[0072] In some embodiments, the proteins disclosed herein are fused or conjugated to a moiety that provides half-life extension ("HLE"). HLE can be achieved via PEGylation or through alternative mechanisms, such as, but not limited to, the addition of an albumin binding domain. In some embodiments, the proteins disclosed herein do not include an Fc fragment, but are fused or conjugated to a moiety that provides HLE. In some embodiments, the moiety that provides HLE is fused to the heavy chain. In some embodiments, the moiety that provides HLE is fused to the light chain.

[0073] The proteins disclosed herein include antibody molecule domains and regions. The term "antibody" generally refers to an immunoglobulin (Ig) molecule that comprises four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential target binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0074] In full-length antibodies, each heavy chain comprises a heavy chain variable domain (abbreviated herein as VH domain) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. IgG, IgA and IgD constant regions comprise a flexible hinge region between the CH1 and CH2 domains. Each light chain comprises a light chain variable domain (abbreviated herein as VL domain) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL domains can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). Each VH and VL domain is composed of three CDRs and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0075] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is usually defined as extending from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The numbering of residues in the Fc region is according to the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. The Fc region may exist in a dimeric or monomeric form. The Fc region binds to various cellular receptors (e.g., Fc receptors) and other immune molecules (e.g., complement proteins).

[0076] The proteins provided herein contain two Fab fragments, which are monovalent antigen-binding fragments consisting of the VL, VH, CL and CH1 domains.

[0077] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2) or subclass. IgG, IgD, and IgE antibodies generally contain two identical heavy chains and two identical light chains and two antigen-binding domains, each composed of a VH and a VL. Generally, IgA antibodies are composed of two monomers, each composed of two heavy chains and two light chains (for IgG, IgD, and IgE antibodies); thus, IgA molecules have four antigen-binding domains, each also composed of a VH and a VL. Certain IgA antibodies are monomeric antibodies in that they are composed of two heavy chains and two light chains. Secretory IgM antibodies are generally composed of five monomers, each composed of two heavy chains and two light chains (as for IgG and IgE antibodies). Thus, the IgM molecule has 10 antigen-binding domains, each also composed of a VH and a VL. The cell surface form of IgM has a two heavy chain / two light chain structure similar to IgG, IgD and IgE antibodies.

[0078] As used herein, the terms "immunological binding" and "immunological binding properties" refer to non-covalent interactions of the type that occur between an immunoglobulin molecule (e.g., an antibody or antigen-binding portion thereof), or a protein that contains an immunoglobulin-derived binding domain(s), and an antigen for which the immunoglobulin or protein is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ), where K dA smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, where the rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, the "on rate constant" (K on ) and "off rate constant" (K off Both K and K can be determined by calculation of the concentrations and the actual rates of association and dissociation (see Malmqvist, Nature 361:186-187 (1993)). off / K on The ratio of d (See Davies et al. (1990) Annual Rev Biochem 59:439-473). The antibodies or antigen-binding portions provided herein have an equilibrium binding constant (K d An antibody is said to specifically bind PD-L1 or CD3 when its K is ≦10 μM, preferably ≦10 nM, more preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM. d One method for determining is by using surface plasmon resonance (SPR), typically by using a biosensor system such as a Biacore® system.

[0079] Functionally, the binding affinity of the proteins provided herein is greater than or equal to 10 -5 M~10 -12 For example, the binding affinity of the proteins provided herein can be in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10-12 M、10 -9 M~10 -12 M、10 -5 M~10 -11 M、10 -6 M~10 -11 M、10 -7 M~10 -11 M、10 -8 M~10 -11 M、10 -9 M~10 -11 M、10 -10 M~10 -11 M、10 -5 M~10 -10 M、10 -6 M~10 -10 M 、 10 -7 M~10 -10 M、10 -8 M~10 -10 M、10 -9 M~10 -10 M、10 -5 M~10 -9 M、10 -6 M~10 -9 M、10 -7 M~10 -9 M、10 -8 M~10 -9 M、10 -5 M~10 -8 M、10 -6 M~10 -8 M、10 -7 M~10 -8 M、10 -5 M~10 -7 M、10 -6 M~10 -7 Mまたは10 -5 M~10 -6 Mである。

[0080] Provided herein are proteins comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, where the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) Domain, a first CH1 Domain, a first linker, an anti-CD3 VH Domain, and a second CH1 Domain; and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL Domain, and a second immunoglobulin light chain constant region.

[0081] Provided herein are proteins comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, where the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) Domain, a first CH1 Domain, a first linker, an anti-CD3 light chain variable (VL) Domain, and a first immunoglobulin light chain constant region, and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL Domain, a second immunoglobulin light chain constant region, a second linker, an anti-CD3 VH Domain, and a second CH1 Domain.

[0082] In some embodiments, the heavy chain comprises, in order from N-terminus to C-terminus, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0083] In some embodiments, the protein further comprises a third polypeptide chain comprising a hinge and an Fc region. In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124.

[0084] Provided herein are proteins comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, where the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, a second CH1 domain, a first hinge, a first CH2 domain, and a first CH3 domain; and the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL domain, a second immunoglobulin light chain constant region, a second hinge, a second CH2 domain, and a second CH3 domain.

[0085] In some embodiments, the first linker comprises an amino acid sequence of any one of SEQ ID NOs: 1-12. In some embodiments, the first linker comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-12.

[0086] In some embodiments, the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 1-12. In some embodiments, the second linker comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of any one of SEQ ID NOs: 1-12.

[0087] In some embodiments, the first linker comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 12, and the second linker comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 12. In some embodiments, the first linker comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 12, and the second linker comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 12.

[0088] In some embodiments, the first linker is the same as the second linker. In some embodiments, the first linker is not the same as the second linker.

[0089] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 50. In some embodiments, the anti-PD-L1 VH domain comprises an HCDR2 comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-PD-L1 VH domain comprises an HCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0090] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0091] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; the anti-PD-L1 VL domain comprises at least one LCDR sequence selected from an LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0092] In some embodiments, the anti-PD-L1 VH domain comprises at least one HCDR sequence selected from HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, HCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; the anti-PD-L1 VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0093] In some embodiments, the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0094] In some embodiments, the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; and the anti-CD3 VL domain comprises at least one LCDR sequence selected from an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0095] In some embodiments, the anti-CD3 VH domain comprises at least one HCDR sequence selected from HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; and the anti-CD3 VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 32.

[0096] In some embodiments, the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0097] In some embodiments, the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; and the anti-CD3 VL domain comprises at least one LCDR sequence selected from an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 32.

[0098] In some embodiments, the anti-CD3 VH domain comprises at least one HCDR sequence selected from HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; the anti-CD3 VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 32.

[0099] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:20, an HCDR2 comprising the amino acid sequence of SEQ ID NO:21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:23, an LCDR2 comprising the amino acid sequence of SEQ ID NO:24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:25; the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; and the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0100] In some embodiments, the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:20, an HCDR2 comprising the amino acid sequence of SEQ ID NO:21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:23, an LCDR2 comprising the amino acid sequence of SEQ ID NO:24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:25; the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:29; and the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:30, an LCDR2 comprising the amino acid sequence of SEQ ID NO:31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:32.

[0101] Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) Domain, a first CH1 Domain, a first linker, an anti-CD3 VH Domain, and a second CH1 Domain; the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL Domain, and a second immunoglobulin light chain constant region; the anti-PD-L1 VH Domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:20, an HCDR2 comprising the amino acid sequence of SEQ ID NO:21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:22; the anti-PD-L1 VL Domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:23, an LCDR2 comprising the amino acid sequence of SEQ ID NO:24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:25; The VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; the first linker comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 12; and the second linker comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 12.

[0102] Provided herein is a protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, wherein the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) Domain, a first CH1 Domain, a first linker, an anti-CD3 VH Domain, and a second CH1 Domain; the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) Domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL Domain, and a second immunoglobulin light chain constant region; the anti-PD-L1 VH Domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:20, an HCDR2 comprising the amino acid sequence of SEQ ID NO:21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:22; the anti-PD-L1 VL Domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO:23, an LCDR2 comprising the amino acid sequence of SEQ ID NO:24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:25; The VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; the first linker comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 12; and the second linker comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 12.

[0103] In some embodiments, the anti-PD-L1 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 33, and 54-107. In some embodiments, the anti-PD-L1 VH domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 33, and 54-107.

[0104] In some embodiments, the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-PD-L1 VL domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:34.

[0105] In some embodiments, the anti-PD-L1 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 33 and 54-107, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 34.

[0106] In some embodiments, the anti-CD3 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 44-48. In some embodiments, the anti-CD3 VH domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 44-48.

[0107] In some embodiments, the anti-CD3 VL domain comprises the amino acid sequence of any one of SEQ ID NOs: 37-42. In some embodiments, the anti-CD3 VL domain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 37-42.

[0108] In some embodiments, the anti-CD3 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 44-48, and the anti-CD3 VL domain comprises the amino acid sequence of any one of SEQ ID NOs: 37-42.

[0109] In some embodiments, the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 116-123, 129-132, 137-141, 142, 144, 146, 147, 148, 150, and 152. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 116-123, 129-132, 137-141, 142, 144, 146, 147, 148, 150, and 152.

[0110] In some embodiments, the light chain comprises the amino acid sequence of any one of SEQ ID NOs: 53, 108-115, 125-128, and 133-136, 143, 145, 151, and 153. In some embodiments, the heavy chain comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NOs: 53, 108-115, 125-128, and 133-136, 143, 145, 151, and 153.

[0111] In some embodiments, (a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 122, the light chain comprises the amino acid sequence of SEQ ID NO: 114, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (b) the heavy chain comprises the amino acid sequence of SEQ ID NO: 123, the light chain comprises the amino acid sequence of SEQ ID NO: 115, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (c) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (d) the heavy chain comprises the amino acid sequence of SEQ ID NO: 117, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (e) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (f) the heavy chain comprises the amino acid sequence of SEQ ID NO: 119, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (g) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (h) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (j) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (k) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (l) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (m) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (n) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 112, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (o) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 113, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (p) the heavy chain comprises the amino acid sequence of SEQ ID NO: 121, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (q) the heavy chain comprises the amino acid sequence of SEQ ID NO: 131, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; or (r) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; Proteins are provided herein.

[0112] Further provided herein are proteins LB201, LB202, LB203, LB204, LB205, LB206, LB207, LB208, LB209, LB210, LB211, LB212, LB213, LB214, LB215, LB216, LB217, LB218, LB219, LB220, LB221, LB222, LB223, LB224, LB225, LB226, LB227, LB228, LB229, LB230, LB231, LB232, LB233, LB234, LB235, LB236, LB237, LB238, LB239, LB240, LB241, LB242, LB243, and LB244. The sequences of the polypeptide chains of these proteins are provided in Table 9. [Table 9-1] [Table 9-2] [Table 9-3]

[0113] Also provided herein is a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence provided herein with one, two or three conservative amino acid substitutions; and the light chain comprises the amino acid sequence provided herein with one, two or three conservative amino acid substitutions.In some embodiments, conservative amino acid substitutions are only made in FR sequence, and not in CDR sequence.In some embodiments, conservative amino acid substitutions are not made in the first linker sequence or the second linker sequence.

[0114] In some embodiments, the proteins provided herein comprise an immunoglobulin heavy chain constant region at the C-terminus of the heavy chain. In some embodiments, the proteins provided herein comprise an immunoglobulin heavy chain constant region at the C-terminus of both the first polypeptide chain and the second polypeptide chain. In some embodiments, the immunoglobulin heavy chain constant region is IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the immunoglobulin heavy chain constant region is IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the immunoglobulin heavy chain constant region is IgG1. In some embodiments, the immunoglobulin heavy chain constant region is immunologically inert. In some embodiments, the immunoglobulin heavy chain constant region comprises one or more mutations to reduce or prevent FcγR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the immunoglobulin heavy chain constant region is a wild-type human IgG1 constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S, or a human IgG4 constant region comprising the amino acid substitution S228P, where the numbering is according to the EU index as in Kabat. In some embodiments, the amino acid residue positions in the constant region of an immunoglobulin molecule are numbered according to the EU index as in Kabat (Ward et al., 1995 Therap. Immunol. 2:77-94).

[0115] When the protein provided herein comprises CH2 and CH3 regions on two polypeptide chains, the CH2 or CH3 region may comprise a site for aiding pairing of the two polypeptide chains.Any suitable Fc heterodimerization technique may be used for pairing of the polypeptide chains.In some embodiments, N-linked glycosylation site is comprised in the CH2 region.In some embodiments, knob and hole mutation is comprised in the CH3 region.

[0116] In some embodiments, the proteins provided herein can comprise an immunoglobulin light chain constant region that is a kappa light chain. In some embodiments, the kappa light chain comprises SEQ ID NO: 15.

[0117] In some embodiments, the proteins provided herein can include an immunoglobulin light chain constant region that is a lambda light chain.

[0118] In some embodiments, the protein provided herein may comprise an immunoglobulin heavy chain constant region comprising the amino acid sequence of a human IgG4, human IgG4(S228P), human IgG2, human IgG1, or human IgG1 effector null Fc region. For example, the human IgG4(S228P) Fc region comprises the following substitutions compared to a wild-type human IgG4 Fc region: S228P. For example, the human IgG1 effector null Fc region comprises the following substitutions compared to a wild-type human IgG1 Fc region: L234A, L235A, and G237A. In some embodiments, the protein may comprise an immunoglobulin heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In some embodiments, the protein may comprise an immunoglobulin heavy chain constant region comprising an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0119] In some embodiments, the protein may include a hinge (e.g., an Fc hinge) that is a wild-type human IgG1 hinge, a wild-type human IgG2 hinge, a wild-type human IgG3 hinge, or a wild-type human IgG4 hinge. In some embodiments, the protein may include an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of a wild-type human IgG1 hinge, a wild-type human IgG2 hinge, a wild-type human IgG3 hinge, or a wild-type human IgG4 hinge. In some embodiments, the protein may include a hinge that includes an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the hinge sequence in any one of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0120] Provided herein are immunoconjugates comprising a protein disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an antiangiogenic agent, an antiproliferative agent, or a proapoptotic agent.

[0121] Examples of suitable therapeutic agents include, but are not limited to, immunomodulators, cytotoxins, radioisotopes, chemotherapeutic agents, antiangiogenic agents, antiproliferative agents, proapoptotic agents, and cytostatic and cytolytic enzymes (e.g., RNAses).Additional therapeutic agents include therapeutic nucleic acids (e.g., genes that code for immunomodulators, antiangiogenic agents, antiproliferative agents, or proapoptotic agents).These drug descriptors are not mutually exclusive, so therapeutic agents can be described using one or more of the above terms.

[0122] Examples of therapeutic agents suitable for use in immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansines, CC-1065 and duocarmycins, calicheamicins and other enediynes, and auristatins. Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers may also be used in immunoconjugates. In addition, conjugates may be made using a secondary carrier (e.g., liposomes or polymers) as the cytotoxic agent. Suitable cytotoxins include agents that inhibit or prevent the function of cells and / or cause the destruction of cells. Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins (e.g., protein kinases, phosphatases, topoisomerases, enzymes, and cyclins).

[0123] Representative cytotoxins include doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pirarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacytidine, doxifluridine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, adriamycin, cisplatin ... These include, but are not limited to, platins, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, fluorouracils, etoposide, taxol, taxol analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramicin, azaserine, bleomycin, tamoxifen, idarubicin, dolastatins / auristatins, hemiasterlin, esperamicin, and maytansinoids.

[0124] Suitable immunomodulatory agents include anti-hormonal agents that block hormone action on the tumor and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0125] Pharmaceutical Compositions The activatable proteins provided herein (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the protein (or an immunoconjugate comprising the protein) and a pharma- ceutically acceptable carrier. In some embodiments, such compositions typically include the protein (or an immunoconjugate comprising the protein) and a pharma- ceutically acceptable carrier, diluent or excipient. Such substances should be non-toxic and should not interfere with the efficacy of the protein. The exact nature of the carrier or other substance will depend on the route of administration, which may be by injection, bolus, infusion, or any other suitable route, as discussed below.

[0126] As used herein, the term "pharmaceutical acceptable" refers to molecular entities and compositions that generally do not produce allergic reactions or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions that have been approved by a regulatory agency of the U.S. Federal or state government or are listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, and more particularly in humans, are considered to be "pharmaceutical acceptable". As used herein, the term "pharmaceutical acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference in the field, which is incorporated herein by reference. Some examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles (e.g., fixed oils) may be used. The use of such media and agents for pharma-ceutically active substances is well known in the art. Except where any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds may also be incorporated into the composition. A pharma-ceutically acceptable carrier, diluent or excipient may be a compound or combination of compounds that does not cause secondary reactions and can, for example, facilitate the administration of the protein, increase its life span and / or its effectiveness in the body, or increase its solubility in solution.

[0127] The pharmaceutical composition disclosed herein can be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration). The solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent (e.g., water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents); an antibacterial agent (e.g., benzyl alcohol or methylparaben); an antioxidant (e.g., ascorbic acid or sodium hydrogen sulfate); a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)); a buffer (e.g., acetate, citrate, or phosphate), and an agent for adjusting tonicity (such as sodium chloride or dextrose). The pH can be adjusted with an acid or base (e.g., hydrochloric acid or sodium hydroxide). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0128] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (登録商標)(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy needle passage exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent (for example, sugar, polyalcohol such as mannitol, sorbitol, sodium chloride) in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0129] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the ingredients listed above, followed by filtration sterilization if necessary. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required ingredients other than those listed above. In the case of sterile powders and powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze-drying to obtain a powder of the active ingredient plus any additional desired ingredients from its solution that has previously been sterile-filtered.

[0130] Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, troches, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, where the compound in the fluid carrier is applied orally and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or auxiliary substances may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients, or compounds of a similar nature: binders (e.g., microcrystalline cellulose, gum tragacanth, or gelatin); excipients (e.g., starch or lactose), disintegrants (e.g., alginic acid, Primojel, etc.), and / or other suitable agents. (登録商標) , or corn starch); a lubricant (e.g., magnesium stearate); a glidant (e.g., colloidal silicon dioxide); a sweetening agent (e.g., sucrose or saccharin); or a flavoring agent (e.g., peppermint, methyl salicylate, or orange flavoring).

[0131] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0132] Systemic administration can also be by transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated can be used in the formulation.Such penetrants are generally known in the art, and for example, for transmucosal administration, include detergents, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal spray or suppository.For transdermal administration, the active compound can be formulated into ointments, salves, gels, or creams as generally known in the art.

[0133] Medicaments can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0134] In some embodiments, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those of skill in the art. The materials may also be obtained commercially. Liposomal suspensions may also be used as pharma-ceutically acceptable carriers.

[0135] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to a physically separate unit suitable as a unitary dosage for the subject to be treated; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification for dosage unit form of the present invention is determined by and directly depends on the specific characteristics of said active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the art of compounding such active compound for individual treatment.

[0136] In some embodiments, the protein may be provided in lyophilized form for reconstitution prior to administration, for example, lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline solution prior to administration to an individual.

[0137] The pharmaceutical compositions provided herein can be included in a container, pack, or dispenser together with instructions for administration.

[0138] Nucleic acid molecules, vectors, host cells and methods for producing proteins Provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding the amino acid sequence of a protein disclosed herein (or the amino acid sequence of (i) the VH domain, (ii) the VL domain, or (iii) both the VH domain and the VL domain of the protein). Further provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding (i) the heavy chain, (ii) the light chain, or (iii) both the heavy chain and the light chain of a protein disclosed herein. Further provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding (i) the first polypeptide chain, (ii) the second polypeptide chain, or (iii) both the first polypeptide chain and the second polypeptide chain of a protein disclosed herein. In some embodiments, the nucleic acid molecule further encodes a third polypeptide chain (e.g., a third polypeptide chain comprising a hinge, a CH2 domain, and a CH3 domain).

[0139] In some embodiments, a nucleic acid molecule encoding a VH domain, a VL domain, a heavy chain, a light chain, a first polypeptide chain, or a second polypeptide chain comprises a signal sequence (or encodes a leader peptide). In some embodiments, a nucleic acid molecule encoding a VH domain, a VL domain, a heavy chain, a light chain, a first polypeptide chain, or a second polypeptide chain does not comprise a signal sequence (or encodes a leader peptide).

[0140] Also provided herein is an expression vector comprising the nucleic acid molecule described herein. In certain vectors, the nucleic acid molecule is operably linked to one or more regulatory sequences suitable for the expression of the nucleic acid segment in a host cell. In some cases, the expression vector comprises a sequence that mediates replication and comprises one or more selectable markers. As used herein, "vector" refers to a construct that can deliver, and preferably express, one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells (e.g., producer cells).

[0141] Provided herein is a recombinant host cell comprising an expression vector or nucleic acid molecule disclosed herein. A "host cell" includes an individual cell, cell line, or cell culture that can be or has been a recipient for a vector(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. An expression vector can be transfected into a host cell by standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. In some embodiments, a recombinant host cell comprises a single vector or a single nucleic acid molecule encoding both the heavy and light chains of a protein disclosed herein. In some embodiments, a recombinant host cell comprises (i) a first vector or a first nucleic acid molecule encoding the heavy chain of a protein disclosed herein and (ii) a second vector or a second nucleic acid molecule encoding the light chain of a protein disclosed herein.

[0142] The protein molecules of the present invention, or portions thereof, can be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, computational techniques or combinations of such techniques, or other techniques readily known in the art.

[0143] Further provided herein is a method for producing a protein disclosed herein, comprising culturing a recombinant host cell comprising an expression vector as described herein under conditions in which its nucleic acid segment is expressed, thereby producing said protein.The protein can then be isolated from said host cell or culture.Provided herein is a method for producing a protein, comprising culturing a recombinant host cell comprising an expression vector as described herein under conditions in which its nucleic acid molecule is expressed, thereby producing protein; and isolating the protein from the host cell or culture.

[0144] The proteins disclosed herein can be produced by any of a variety of methods known to those skilled in the art. In certain embodiments, the proteins disclosed herein can be recombinantly produced. For example, the nucleic acid sequence encoding one or more of the heavy or light chains provided herein, or a portion thereof, can be introduced into bacterial cells (e.g., E. coli, B. subtilis) or eukaryotic cells (e.g., yeast such as S. cerevisiae, or mammalian cells such as CHO cell lines, various Cos cell lines, HeLa cells, HEK293 cells, various myeloma cell lines, or transformed B cells or hybridomas), or into an in vitro translation system, and the translated polypeptide can be isolated. In some embodiments, the light and heavy chain proteins are produced in cells with signal sequences that are removed during the production of the mature proteins disclosed herein.

[0145] One of skill in the art can determine whether a protein comprising a given polypeptide sequence binds to PD-L1 protein and / or CD3 protein using standard methodologies (e.g., Western blot, ELISA, etc.).

[0146] Medical Uses of Activatable Proteins Provided herein are methods and uses of the activatable proteins, immunoconjugates, and pharmaceutical compositions disclosed herein to provide a therapeutic benefit to a subject with cancer.

[0147] The activatable proteins, immunoconjugates, or pharmaceutical compositions disclosed herein may be used in methods of treatment of the human or animal body, including prophylactic or preventative treatment (e.g., treatment prior to the onset of a condition in a subject, to reduce the risk of the condition occurring in the subject; delay its onset; or reduce its severity after onset). Methods of treatment may include administering the protein, immunoconjugate, or pharmaceutical composition to a subject in need thereof.

[0148] Provided herein is a method for enhancing anti-cancer immune response in a subject, comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.In some embodiments, the anti-cancer immune response is a T cell response.In some embodiments, the anti-cancer immune response is a complement response.

[0149] Provided herein are methods for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0150] Provided herein is a method for ameliorating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0151] Provided herein are methods for reducing tumor size in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0152] Provided herein is a method for inhibiting tumor growth in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0153] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in enhancing an anti-cancer immune response in a subject.

[0154] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in treating cancer in a subject.

[0155] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in ameliorating a symptom of cancer in a subject.

[0156] In some embodiments, the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

[0157] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0158] In some embodiments, the cancer of blood tissue is lymphoma. In some embodiments, the cancer is mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, acute myeloid leukemia (AML), B-lymphoid leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or hairy cell leukemia.

[0159] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical agent (e.g., a protein, immunoconjugate, or pharmaceutical composition disclosed herein) sufficient to reduce or ameliorate the severity and / or duration of cancer, or one or more symptoms thereof, prevent progression of the disease, cause regression of the disease, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disease, or enhance or improve the prophylactic or therapeutic effect(s) of another related treatment (e.g., a prophylactic or therapeutic agent) for cancer.

[0160] The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the administration schedule, and other factors known to medical practitioners. Treatment prescription, such as determining dosage, is within the responsibility of general practitioners and other physicians, and may depend on the severity of symptoms and / or progression of the disease being treated. The appropriate dosage of antibody-based protein molecules is well known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe KD et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages can be found herein or in the Physician's Desk Reference (2003), and can be used as appropriate for the type of medicament being administered. The therapeutically effective amount or appropriate dose of an antibody-based protein molecule can be determined by comparing its in vitro activity and in vivo activity in animal models.Methods for extrapolating effective doses in mice and other test animals to humans are known.The exact dose depends on many factors, including whether the antibody-based protein is for prevention or treatment, the size and location of the area to be treated, the exact nature of the antibody-based protein, and the nature of any detectable label or other molecule that is attached to the antibody-based protein.

[0161] Typical protein doses range from 100 μg to 1 g for systemic application, and 1 μg to 1 mg for intradermal injection. An initial higher loading dose may be administered, followed by one or more lower doses. In some embodiments, the protein is an IgG1 or IgG4 isotype. Doses for single treatment of adult subjects may be adjusted proportionately for children and infants. Treatment may be repeated at daily, twice weekly, weekly or monthly intervals, at the physician's discretion. Treatment schedules for subjects may depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated.

[0162] Treatment may be periodic, with the period between administrations being about 2 weeks or longer, e.g., about 3 weeks or longer, about 4 weeks or longer, about once a month or longer, about 5 weeks or longer, or about 6 weeks or longer. For example, treatment may be every 2-4 weeks or every 4-8 weeks. Treatment may be given before and / or after surgery and / or may be administered or applied directly to the anatomical site of the surgical or invasive procedure. Suitable formulations and routes of administration are described above.

[0163] In some embodiments, the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can be administered as subcutaneous injections, e.g., using an autoinjector for long-term prevention / treatment.

[0164] In some embodiments, the therapeutic effect of a protein, immunoconjugate, or pharmaceutical composition disclosed herein may last for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of a protein, multimeric protein, immunoconjugate, or pharmaceutical composition disclosed herein may last in a subject for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.

[0165] In some embodiments, a subject may be treated with a protein, immunoconjugate, or pharmaceutical composition disclosed herein and an additional therapeutic agent or treatment used to treat cancer or a symptom or complication of cancer. The protein, immunoconjugate, or pharmaceutical composition disclosed herein and the additional therapeutic agent or treatment may be administered simultaneously or sequentially.

[0166] In some embodiments, the subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a guinea pig, a mouse, or a rat. In some embodiments, the subject is an adult. In some embodiments, the subject is a child. In some embodiments, the human subject is 16 years of age or older. In some embodiments, the human subject is 18 years of age or older. In some embodiments, the human subject is under 16 years of age. In some embodiments, the human subject is under 18 years of age.

[0167] Further provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in the treatment of a disease or disorder.

[0168] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use as a medicament. definition

[0169] Unless otherwise noted, the terms used herein have definitions as commonly used in the art. Some terms are defined below, and further definitions can be found within the remainder of the detailed description.

[0170] As used herein, and unless otherwise stated, the terms "hinge linker," "linker," "hinge," "first linker," "second linker," "lower hinge linker" ("LHL"), "inter-Fab linker," and derivatives thereof (in the plural or singular) refer to a sequence that is derived, for example, from an immunoglobulin hinge region and that can link two polypeptides, e.g., polypeptides of different Fab regions, and is separate from any hinge sequence in an immunoglobulin hinge region that may be part of a protein of the invention.

[0171] LB protein refers to a lock body protein of the invention having the structure as defined in FIG.

[0172] The terms "a" or "an" may refer to one or more of its entities, i.e., to a plurality of referents. Thus, the terms "a", "an", "one and more", and "at least one" are used interchangeably herein. Furthermore, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one of said elements is present, unless the context clearly requires that only one such element is present.

[0173] Unless otherwise stated or clear from the context, the term "about" means within 10% above or below the reported numerical value (except when such number is greater than 100% or less than 0% of possible values). When used in conjunction with a range of values ​​or a series of values, the term "about" applies to the endpoints of the range or each of the values ​​recited in that series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0174] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout the range of alignment of residues, e.g., nucleotides or amino acids. The "identity fraction" for an aligned segment of a test sequence and a reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The "percent identity" is the identity fraction x 100. Percent identity can be calculated using the alignment program Clustal Omega (available at ebi.ac.uk / Tools / msa / clustalo) using default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega" (2011 October 11) Molecular Systems Biology 7:539. For purposes of calculating identity to a sequence, extensions (eg, tags) are not included.

[0175] As used herein, the term "HCDR" refers to a heavy chain complementarity determining region. As used herein, the term "LCDR" refers to a light chain complementarity determining region.

[0176] The terms "amino-terminus", "N-terminus", "carboxyl-terminus" and "C-terminus" are used herein to indicate positions within a polypeptide chain. When the context allows, these terms are used in reference to a particular sequence or part of a polypeptide to indicate proximity or relative position. For example, a particular sequence located carboxyl-terminal to a reference sequence within a polypeptide is located close to the carboxyl-terminus of the reference sequence, but is not necessarily at the carboxyl-terminus of the complete polypeptide.

[0177] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another that does not significantly and adversely change the functional activity. A preferred example of a "conservative substitution" is the replacement of one amino acid with another having a value of > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919):

number

[0178] The term "immunoconjugate" refers to a protein of the present disclosure conjugated to a cytotoxic, cytostatic and / or therapeutic agent.

[0179] The term "isolated molecule" (where the molecule is, for example, a protein, nucleic acid, polynucleotide, or antibody) refers to a molecule that, depending on its source of origin or derivative, is (1) not associated with naturally associated components that accompany it in its natural state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecular purity or homogeneity can be assayed by many means well known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution can be provided using HPLC or other means well known in the art for purification.

[0180] The terms "inhibit," "block," or "neutralize," as used herein in reference to the biological activity of a protein disclosed herein, refer to the ability of the protein to substantially antagonize, prohibit, prevent, suppress, slow, destroy, eliminate, halt, reduce, or reverse, for example, the progression, intensity, or severity of what is being inhibited, including, but not limited to, the binding of PD-L1 to PD-1, or the binding of CD3 to the T-cell receptor (TCR).

[0181] As used herein, the terms "treat", "treating" or "treatment of" (and grammatical variations thereof) mean that the severity of the subject's condition is reduced, at least partially ameliorated, or stabilized, and / or some alleviation, reduction, decrease or stabilization of at least one clinical symptom is achieved, and / or there is a delay in the progression of the disease or disorder.

[0182] PD-L1 is also known as programmed cell death ligand 1, CD274, B7-H, B7H1, PDCD1L1, PDCD1LG1, PDL1, and hPD-L1. Exemplary PD-L1 amino acid sequences are provided as SEQ ID NO:35 and SEQ ID NO:36.

[0183] CD3 is also known as cluster of differentiation 3. CD3 is a multimeric protein complex. It is composed of four separate polypeptide chains; epsilon (ε), gamma (γ), delta (δ) and zeta (ζ).

[0184] As used herein, the terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of a disease, disorder and / or clinical symptom(s) in a subject and / or reducing the severity of the onset of a disease, disorder and / or clinical symptom(s) relative to that which would occur in the absence of the compositions and / or methods described herein. Prevention can be complete (e.g., the disease, disorder and / or clinical symptom(s) is / are completely absent). The prevention can also be partial, such that the onset of a disease, disorder and / or clinical symptom(s) in the subject and / or the onset is less severe than that which would occur in the absence of the compositions and / or methods described herein.

[0185] As used herein, a "therapeutically effective amount" is an amount of a protein or pharmaceutical composition provided herein that is effective to treat a disease or disorder or ameliorate a sign or symptom thereof in a subject. A "therapeutically effective amount" may vary depending, for example, on the disease and / or symptoms of the disease, the severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health of the patient to be treated, and the judgment of the prescribing physician.

[0186] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be understood as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0187] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0188] The present disclosure will be further clarified by the following examples, which are merely illustrative of the present disclosure and are in no way intended to be limiting. EXAMPLES

[0189] Working Example Generation of optimized conditionally active therapeutic proteins Introduction In this example, the inventors have successfully generated certain antibodies of the invention, which exhibit restricted binding to a CD3 epitope until at least one lower hinge linker is targeted and cleaved by one or more proteases (e.g., MMPs) that are often found to be highly active in the TME.

[0190] material and method Protein cloning, transient expression, purification, and characterization The DNA sequences encoding the polypeptides were cloned into expression cassettes encoding separate human IgG1 heavy and light chain constant regions in separate plasmid vectors via restriction-ligation cloning to generate activatable constructs for expression.The proteins were expressed in CHO cells and purified from culture supernatants via a combination of Protein A affinity chromatography (ProA), ion exchange chromatography (IEX) and / or size exclusion chromatography (SEC).The purified proteins were characterized by SEC, SDS-PAGE and mass spectrometry.

[0191] Metalloprotease digestion Protein constructs were incubated with human matrix metalloprotease (MMP) enzyme MMP12 at a ratio of 1% total MMP to protein construct (wt / wt) in Tris-buffered saline (pH 7.4) containing 5 mM CaCl2 for time increments between 0 and 24 hours at 37° C. The reaction was stopped by the addition of 20 mM EDTA and samples were then tested for binding or functional activity as described.

[0192] T cell activation bioassay Functional activity of protein constructs was evaluated in a co-culture assay using MDA-MB-231 (PD-L1 high) or A549 cells (PD-L1 low) with NFAT-RE-luciferase Jurkat reporter cell line (Promega - TCR / CD3 Effector cells NFAT). MDA-MB-231 (PD-L1 high) or A549 cells (40000 cells / well) were seeded into 96-well white clear bottom tissue culture treated plates in Hybri-Care medium (ATCC) supplemented with 10% FBS and incubated overnight at 37°C in a CO2 incubator. The medium was removed and control antibodies or protein constructs (± MMP3 / 7 / 12 pre-digested) prepared in assay medium (RPMI supplemented with 10% FBS) were added to the cells. TCR / CD3 effector cells (NFAT) were thawed, diluted according to the manufacturer's protocol, and then added to the assay wells. After 6 hours of incubation in a CO2 incubator at 37°C, the plates were re-equilibrated to room temperature and luciferase activity was determined by adding Bio-Glo reagent for 5-10 minutes and measuring the luminescence signal (RLU). Fold induction was determined by calculating the ratio of sample RLU / RLU in the absence of antibody after subtraction of the background luminescence signal.

[0193] ELISA binding of IgG and LB proteins For ELISA binding assay, 384-well or 96-well plates were coated with 1 μg / ml and incubated overnight at 4° C. and protected from light. Plates were washed twice with PBS / 0.05% Tween® 20 and then blocked with blocking buffer (3% milk protein in PBS) for 1 hour at room temperature. Test agents or controls were then loaded and incubated for 1 hour at room temperature. Plates were then washed three times with PBS / 0.05% Tween® 20. Finally, HRP-conjugated secondary antibodies (diluted 1 / 5,000 in blocking buffer) were added and incubated for 1 hour at room temperature. Plates were washed again three times with PBS / 0.05% Tween®, after which TMB substrate was added and incubated for 6 minutes at room temperature. The reaction was subsequently stopped with stop solution. Absorbance was read at 450 nm and 570 nm. The absorbance at 570 nm was subtracted from the absorbance at 450 nm for each plate.

[0194] Cell binding of LB proteins Jurkat cells were harvested, counted, washed with PBS, and then stained with Zombie UV viability dye diluted 1000-fold in PBS for 30 min at room temperature (RT). Induction was determined by calculating the ratio of sample RLU / RLU in the absence of antibody after subtraction of background luminescence signal. Cells were washed with staining buffer (0.1% BSA in PBS), suspended in staining buffer, and divided into test aliquots. Cells were incubated with LB protein for 30 min on ice at the indicated times. Cells were washed twice with staining buffer and then incubated with secondary antibody or staining buffer alone for 60 min on ice. After incubation with secondary antibody, cells were washed twice with staining buffer and then fixed with 4% PFA for 10 min at RT. Fixed cells were resuspended in staining buffer and stored at 4° C. before analysis using a BD LSR Fortessa flow cytometer. To test PD-L1 expression on A549, MDA-MB-231 and RKO cells, cells were plated and incubated with 20ng / ml IFNγ for 45 hours, then washed once with PBS and resuspended in 1ml TrypLE. TM The cells were then detached with Express Enzyme for 5 min at 37°C in a standard incubator. TM Fixable Green (=FITC) was used to stain for viability and incubated for 20 min in the refrigerator. Labeled clone MIH2 (to detect PD-L1 expression) or isotype IgG was incubated with the viability stained cells for 30 min at 4° C. Samples were centrifuged, washed twice and then measured on a BD FACS Canto II.

[0195] Surface plasmon resonance of Fab proteins To assess the binding of the test Fabs to human CD3 (Acro Biosystems, Newark, USA) and cynomolgus monkey CD3 (Acro Biosystems, Newark, USA), multicycle kinetic analysis was performed using a Biacore ELISA kit at 25°C.(登録商標) 8K (serial no. 2724204) and HBS-P+ (Cytiva, Marlborough, USA) supplemented with 0.1% BSA (Sigma, Dorset, UK) was used as running buffer and for ligand and analyte dilution. Fab samples were diluted to 1.0 μg / mL in running buffer and at the start of each cycle were F-coupled to a Series S CM5 chip (Cytiva, Marlborough, USA) previously coupled with an anti-human Fab capture antibody (Cytiva, Marlborough, USA) using standard amine chemistry. c The ligand was captured at a flow rate of 10 μl / min, resulting in an immobilization level (R L The surface was then stabilized. Multi-cycle kinetic data was analyzed to minimize any potential mass transfer effects. (try and minimize) The data were acquired using either human or cynomolgus proteins as analytes injected at a flow rate of 30 μl / min. An eight-point two-fold dilution range from 100.0 nM to 0.78 nM was prepared in running buffer for the antigen. For each concentration, the association phase was monitored for 240 s and the dissociation phase was measured for 600 s. Regeneration of the sensor chip surface was performed between cycles using 10 mM glycine (pH 2.1). Multiple repeats of blank and antigen were programmed into the kinetic run to check the stability of both the surface and the analyte over the kinetic cycles. Reference F c The signal from 1 (no ligand captured) was multiplied by F to correct for bulk effects and differences in non-specific binding to the reference surface. cThe signal from each blank run (ligand captured but not antigen) was subtracted to correct for differences in surface stability. Binding was analyzed using 1:1 binding analysis due to the high affinity interaction between antibody and antigen. The data were analyzed using a procedure called double referencing, which first measures the response of the reference channel (F ) to compensate for bulk effects, baseline drift, and small differences between the reference and active channels. c 1) and then subtracting the zero concentration sensorgram.

[0196] Primary T cell killing against A549, MDA-MB-231, and RKO The respective tumor cells were counted and seeded into appropriate 96-well plates and allowed to adhere for approximately 24 hours. PBMC cells were isolated from whole blood by centrifugation, followed immediately by T cell isolation. For this purpose, PBMC were incubated with Pan T cell MicroBead cocktail and subsequently loaded onto a MACS separator column. The flow-through cells, representing enriched T cells, were collected and diluted to the desired level. For the Incucyte® live cell analysis platform, LB protein or a buffer with an appropriate concentration of Annexin V dye was added to the adherent tumor target cells, followed immediately by the addition of T cells and the plate was placed in the Incucyte® S3 Live-Cell Analysis System incubator. The scan interval was set to every 4 hours for 72-96 hours. Annexin V data was normalized to the buffer / T cell / tumor cell signal before plotting. In vivo evaluation Humanized mice were generated by myeloablation, transplantation of hCD34+ HSC into NCG mice, and subsequent myeloid cytokine boost. MDA-MB-231 TNBC cells were transplanted, resulting in a mean tumor volume of 80 mm 3Tumors were allowed to grow to 100% sigma-free media. Mice were then randomized and treated with either IgG isotype control antibody, atezolizumab, or LB protein at the indicated doses by intraperitoneal injection every 3 days for up to 8 times. Tumor sizes were measured using calipers every 3 or 4 days, and average values ​​were plotted using Graphpad Prism.

[0197] Results and Discussion In silico affinity maturation of anti-PD-L1 binding to human and cynomolgus PD-L1 The sequences of human and cynomolgus monkey (cyno) PD-L1 proteins are provided in Table 10. [Table 10]

[0198] Inspection of the crystal structure of human PD-L1 (PDB: 5GGT) in complex with the Fab domain of the anti-PD-L1 antibody (BMS-936559) disclosed in U.S. Patent No. 7,943,743 B2, and a model of the cyno PD-L1 equivalent showed that the only differences between the human and cyno PD-L1 epitopes are Ala51Thr Ala52Ser, as shown in Alignment Table 11. [Table 11] huPD-L1_19-127=SEQ ID NO: 35 cynoPD-L1 = SEQ ID NO: 36

[0199] Furthermore, Ala51Thr and Ala52Ser interact primarily with the VH backbone and not with amino acid side chains. Taken together, these facts made it difficult to differentially affect BMS-936559 binding to human and cyno PD-L1. As a result, in silico saturation mutagenesis was performed on VH residues proximal to the Ala51Thr Ala52Ser site, or elsewhere on the VH paratope, using Rosetta and MOE software to attempt to equalize the binding affinity of BMS-936559 to human and cyno PD-L1. A negative (favorable) ddG affinity for PD-L1 was calculated for the variant, and mutations were selected if they did not change or only slightly improved the ddG affinity for that same variant binding to human PD-L1. A set of 54 variants spanning 13 residue positions in the paratope of the VH of BMS-936559 with PD-L1 was generated, as shown in Table 6. These 54 variants were produced as Fab proteins by transient transfection of CHO cells and then purified using CH1 affinity purification. After purification, homogeneity was assessed by analytical SEC (Table 12) and proteins with purity >87% were further evaluated for binding to human and cyno PD-L1 in SPR (Table 13). [Table 12-1] [Table 12-2] [Table 12-3]

[0200] Table 13 shows the SPR binding results of selected anti-PD-L1 variants to human and cyno PD-L1 proteins. WT affinity was determined for human PD-L1 with a KD(M) of 4.4E-10, while cyno PD-L1 affinity is approximately 10-fold lower with a KD(M) of 7.9E-9. Mut-6, -23, -43 showed increased affinity for human and cyno PD-L1, while Mut-50 showed increased affinity only for human PD-L1. Mut-48 and Mut-49 showed improved affinity for cyno PD-L1 but not for human PD-L1 compared to WT. [Table 13-1] [Table 13-2] ND = no binding detected

[0201] LB protein construct design, cloning, expression, and characterization To generate proteins for functional testing, DNA cassettes for each construct type were designed using combinations of anti-PD-L1 variable domain, constant domain and linker sequences found in Table 1 in combination with mouse or humanized variable domain variants of the anti-CD3 antibody SP34 (as shown in Table 2). To ensure the suitability of the SP34-derived domains with respect to the structural format of these proteins, a series of novel SP34 humanized variants were tested alongside known SP34 V domain sequences in mouse and humanized forms as controls (Table 2). These humanized variants were then combined with PD-L1 variable domain, constant domain and linker to form full-length heavy and light chain sequences (Table 3). Using these full-length chains, 16 initial designs were synthesized and cloned into expression vectors encoding human IgG1-based heavy and light chain sequences and free hinge-Fc fragments (Table 4). The anti-PD-L1 variable domain sequences used in the protein constructs disclosed herein are the variable domain sequences provided in U.S. Patent No. 7,943,743 B2 and Tables 5 and 6. Proteins were produced by transient transfection of CHO cells and then purified by proA, IEX and / or SEC. After the ProA step, proteins were examined for yield and homogeneity by SEC (Table 14). Fully purified proteins showed high purity (>95%) and homogeneity by analytical SEC. This indicates that the best-behaved constructs can be expressed and purified in a single process as intact and stable products. [Table 14] HMW = high molecular weight product LMW=low molecular weight product

[0202] In vitro functional characterization Purified proteins with higher yield and / or homogeneity characteristics in Table 13 (LB204, LB205, LB206, LB208, LB209, LB210, and LB213) were incubated with or without human MMP12 enzyme for 0 or 4 hours at 37° C. FIG. 5A shows the digestion profile of exemplary LB proteins (LB204, 206, 208, 209, 210, 213, and 220). The intact proteins showed the expected banding pattern in reducing and non-reducing SDS PAGE, with one predominant band visible at 150 kDa in the non-reducing gel, and three chains visible at 75 kDa (LB heavy chain), 50 kDa (LB light chain), and approximately 30 kDa (Fc base) in the reducing gel. Upon incubation with MMP12 at 37°C, striking differences in band distribution compared to the intact protein suggested cleavage of both the LB heavy and LB light chains. In reducing SDS-PAGE, the appearance of multiple 25-30 kDa bands (corresponding to cleaved VC domains from either the LB light or heavy chains and single chain Fc fragments released by proteolysis) and a ∼55 kDa band (corresponding to the VH-CH domains with Fc) and the corresponding disappearance of intact LB chain and heavy chain products at 75 and 50 kDa indicated cleavage of either intact chain at both of the linkers, thereby creating the respective products as shown in Figure 1A. Selected LB proteins were then tested for CD3 activation signals in mixed cell culture assays using a human PD-L1+ cell line, MDA-MB-231, and a Jurkat cell line engineered to provide a reporter signal for human CD3 activation (Figures 3A-G). This assay therefore tests the ability of molecules to bind the cell surface of the MDA-MB-231 cell line (through PD-L1 for the test article, or through Her2 for the BiTE control protein) and activate CD3 through trans-presentation of the CD3 binding domain in a Jurkat reporter line.The Her2 / CD3 BiTE positive control protein induced strong, concentration-dependent CD3 activation, whereas the IgG1 isotype negative control did not induce any signal. All test article samples showed low or no CD3 activation signal at 0 hours MMP12 incubation (i.e. intact uncleaved protein). This indicates that the CD3-binding SP34 domain minimizes the ability to bind CD3 in all constructs. However, unexpectedly, only proteins LB206 and LB213 showed high concentration-dependent activation at 4 hours with the desired combination of low / no CD3 signal at 0 hours. Indeed, construct LB210 showed no signal after activation (Figures 3A-G). These findings indicated that both the linker type and the humanized sequence of the CD3 variable domain are crucial factors in the performance of the resulting molecules.

[0203] The anti-CD3 SP34 humanized V domain variants contained in LB206 and LB213 were then further characterized in Fab and IgG formats. First, surface plasmon resonance (SPR) of the purified Fab proteins was used to confirm binding to commercially available human and cyno CD3δε heterodimer peptides (Figures 4A and B). D The values ​​were calculated to be 29 nM and 46 nM, respectively, although the off-rate of anti-CD3-Fab-001 was significantly different from that of anti-CD3-Fab-002 against human CD3δε heterodimers (4.7 × 10 -3 vs. 2.0x10 -2). In contrast, purified IgGs of CD3-IgG00-1 and CD3-IgG-002 did not show any apparent affinity difference in binding ELISA to the same commercially available human and cyno CD3δε heterodimer proteins (Figure 4C). Finally, purified IgG proteins were tested for CD3 activation signal in a Jurkat cell line engineered to provide a reporter signal of human CD3 activation (Figure 4D). This assay tested the ability of the new humanized variants to activate CD3 in a Jurkat reporter cell line in direct comparison with the mouse SP34 IgG molecule. These findings showed that anti-CD3-IgG-001 showed similar potency to SP34 in the Jurkat reporter cell line (EC50 for both molecules in this assay was 0.2 nM), whereas anti-CD3-IgG-002 showed an approximately 5-fold lower apparent EC50 in this assay (EC50 0.9 nM). Taken together, these results showed that the CD3-dependent agonist properties of SP34 were preserved in the anti-CD3-IgG-001 humanization, whereas anti-CD3-IgG-002 showed a lower K observed in the SPR analysis. D And consistent with the faster Kd kinetics, it displayed a 5-fold lower agonistic potency in Jurkat reporter cells.

[0204] Having identified LB206 and LB213 as functional molecules with the desired activity, further proteins were designed based on these for expression and subsequent functional testing. As before, DNA cassettes for each construct type and different modalities (Figure 1B) were designed using combinations of variable domains, constant domains and linker sequences found in Table 1. The proteins were produced by transient transfection of CHO cells and then purified by ProA, IEX and / or SEC. After the ProA step, the proteins were examined for yield and homogeneity by SEC (Table 15). Significant differences in yield were observed for some modalities over others, but all fully purified proteins showed high purity (>95%) and homogeneity by analytical SEC. This indicates that the best-behaved constructs can be expressed and purified in a single process as intact and stable products. [Table 15-1] [Table 15-2] HMW = high molecular weight product LMW=low molecular weight product ND = No Data

[0205] Selected molecules were again incubated in the presence or absence of human MMP12 enzyme at 37° C. for the indicated times, and digestion profiles were assessed using SDS-PAGE under reducing and non-reducing conditions (FIGS. 5A and 5B). FIG. 5A shows the digestion profiles of exemplary LB proteins (LB204, 206, 208, 209, 210, 213, 220). The intact protein showed the expected banding pattern in reducing and non-reducing SDS PAGE, with one predominant band visible at 150 kDa in the non-reducing gel, and three chains visible at 75 kDa (LB heavy chain), 50 kDa (LB light chain) and approximately 30 kDa (Fc base) in the reducing gel (FIG. 5A). Upon incubation with MMP12 at 37° C., the striking difference in band distribution compared to the intact protein suggested cleavage of both the LB heavy and LB light chains. In reducing SDS-PAGE, the appearance of multiple 25-30 kDa bands (corresponding to cleaved VC domains from either the LB light or heavy chains, and the single chain Fc fragments released by proteolysis) and a band at approximately 55 kDa (corresponding to the VH-CH domains with Fc) and the corresponding disappearance of the intact LB and heavy chain products at 75 and 50 kDa indicate cleavage of either intact chain at both co-linkers, thereby creating the respective products as shown in Figure 1A. Further enzymatic digestion and SDS-PAGE analysis (Figure 5B) showed differences in the rate at which the different linker designs were cleaved by MMP12. For example, comparison of LB206 and LB220 in this manner showed a rapid disappearance of the intact heavy chain of LB220 within 15 min, while a significant amount remained in the LB206 digest. This suggests that the LHL linker present in LB220 is more susceptible to MMP12 cleavage than the LHL linker present in LB206.

[0206] Additionally, several additional exemplary LB proteins differing in LHL linker and CD3 V domain combinations were selected based on protein expression yield, final purity, and homogeneity. Parallel digestion profiles were compared in screening assays for functionality. To this end, LB217, LB218, and LB220 were tested for CD3 activation signals in mixed cell culture assays using a human PD-L1+ cell line, MDA-MB-231, and a Jurkat cell line engineered to provide a reporter signal for human CD3 activation (Figures 6A-6C). All test items showed low CD3 activation signals at 0 hours MMP12 incubation time (i.e., intact uncleaved protein), while the IgG1 isotype negative control did not induce any signal. This indicates that the CD3-binding SP34 domain has minimal ability to bind CD3 in all constructs. Consistent with the previously observed digestion profile of LB220, the luciferase signal in Jurkat reporter cells disappeared rapidly with increasing MMP12 incubation time (0.5 h vs. 1 h) compared to LB217 (Figure 6A) and LB218 (Figure 6B) (Figure 6C). Furthermore, LB217, which contains a human CD3 variable domain based on LB206, showed the highest maximal signal compared to the LB213-based CD3 variable domain-containing proteins LB218 and LB220. Taken together, these findings indicate and confirm that both the linker type and the humanized sequence for the CD3 variable domain are crucial factors in the performance and activation profile of the resulting LB molecule.

[0207] To further evaluate the ability of undigested, fully intact, and MMP12-incubated proteins to interact with CD3 and PD-L1, binding ELISAs to recombinant human and cyno CD3δε heterodimers, as well as huPD-L1 domains, were performed with exemplary proteins (LB206, LB213, LB220; Figure 7). Binding signals for both CD3 orthologues confirmed that CD3 binding is greatly reduced in fully intact proteins, but strongly increases after incubation with MMP12 at 37°C for the times indicated. In contrast, huPD-L1 binding of LB proteins is unaffected by incubation with MMP12 for up to 15 min, but then decreases over time. This is consistent with the cleavage patterns observed in SDS PAGE analysis, which show a clear decrease in the levels of intact heavy and light chains with increasing digestion time, further confirming that both linkers are proteolytically unstable. Specifically, Figure 7A shows ELISA binding of LB206 protein, either intact or incubated with MMP12 for 5 min, to human PD-L1, using atezolizumab as a positive control or IgG1 isotype (no signal) as a negative control. Intact and MMP12-treated LB206 bind to PD-L1 to similar levels. Binding to PD-L1 appears to be lower than atezolizumab, consistent with the one-arm LB protein structure and consistent with the two-arm IgG atezolizumab. Figure 7B shows ELISA binding of LB206 protein, either intact or incubated with MMP12 for 5 min, to human CD3 δε heterodimer or IgG1 isotype (no signal) as a negative control. Intact LB206 binds to human CD3 δε heterodimer at very low levels. In contrast, MMP12-treated LB206 binds strongly to human CD3 δε heterodimer. Figure 7C shows ELISA binding of LB218 protein, either intact or incubated with MMP12 for 5 minutes, 15 minutes, 30 minutes, or 60 minutes, to human PD-L1, using the IgG1 isotype (no signal) as a negative control.Intact and LB218 treated with MMP12 for 5 or 15 min bind to PD-L1 at similar levels. Binding to PD-L1 appears to be low at 30 and 60 min, suggesting the occurrence of protein cleavage 2 (Figure 1A). Figure 7D shows ELISA binding of LB218 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 min, to human CD3 δε heterodimer, with IgG1 isotype (no signal) as a negative control. Intact LB218 shows very low levels of binding to human CD3 δε heterodimer. Higher binding to human CD3 δε heterodimer can be observed with increasing MMP12 treatment time. LB218 treated with MMP12 for 60 min shows the highest binding to human CD3 δε heterodimer. Figure 7E shows ELISA binding of LB218 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 minutes, to cyno CD3 δε heterodimer, using IgG1 isotype (no signal) as a negative control. Intact LB218 shows very low levels of binding to cyno CD3 δε heterodimer. Higher binding to cyno CD3 δε heterodimer can be observed with increasing MMP12 treatment time. LB218 treated with MMP12 for 60 minutes shows the highest binding to cyno CD3 δε heterodimer. Figure 7F shows ELISA binding of LB213 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 minutes, using IgG1 isotype (no signal) as a negative control, to human PD-L1. Intact and LB213 treated with MMP12 for 5 or 15 minutes bind to PD-L1 at similar levels. Binding to PD-L1 appears to be low at 30 and 60 min, suggesting the occurrence of proteolytic cleavage 2 (Figure 1A).FIG. 7G shows ELISA binding of LB213 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 min, to human CD3 δε heterodimer, using IgG1 isotype (no signal) as a negative control. Intact LB213 shows binding to human CD3 δε heterodimer to very low levels. Higher binding to human CD3 δε heterodimer can be observed with increasing MMP12 treatment time. LB213 treated with MMP12 for 60 min shows higher binding to human CD3 δε heterodimer. FIG. 7H shows ELISA binding of LB213 protein, either intact or incubated with MMP12 for 5, 15, 30, or 60 min, using IgG1 isotype (no signal) as a negative control, to cyno CD3 δε heterodimer. Intact LB213 shows binding to cyno CD3 δε heterodimer to very low levels. As the MMP12 treatment time is increased, higher binding to the cyno CD3 δε heterodimer can be observed. LB213 cells treated with MMP12 for 60 minutes show higher binding to the cyno CD3 δε heterodimer.

[0208] Jurkat cell binding assays were then performed on LB206 using flow cytometry, which showed a corresponding binding profile only after MMP12-mediated LB hinge linker digestion (Figure 8). These results indicate that intact LB206 protein has low or negligible binding capacity to CD3+ / PD-L1- cells. They further confirm that the observed low or no activation of luciferase signal in Jurkat reporter cell line-based activation assays of intact / undigested LB protein is due to poor or no presentation of CD3 binding in trans.

[0209] Potency of lead molecules in primary T cell assays To further increase understanding of LB protein functionality, selected LB proteins were evaluated for their ability to induce tumor cell killing by primary T cells. Three representative cancer cell lines were selected as target cells based on their PD-L1 expression levels.

[0210] PD-L1+ low (A549) cells were seeded into 96-well plates and grown for approximately 24 hours before addition of primary T cells (E:T 5:1) from two different donors and intact or digested LB protein (LB206 or LB226) at concentrations ranging from 0.01 nM to 10 nM as indicated. Cancer cell killing was assessed using the Incucyte® Live-Cell Analysis system by measuring total annexin V region over time. Results are shown in Figure 9A. Intact LB206 induced very low level background cell killing at 10 nM, while strong annexin V signal was detected for both 10 nM and 1 nM digested LB206 protein. Cleaved LB206 at 0.1 nM showed very low signal, similar to intact LB206 at 10 nM. This suggests that the ability of undigested LB206 to direct T cell killing is apparently 100-fold lower. Similarly, intact LB218 did not induce A549 cancer cell killing at 10 nM (FIG. 9B). As previously observed with LB206, strong Annexin V signals above background were detected in the 10 nM and 1 nM digested LB218 samples. However, the overall induction of cell killing was lower than that observed with LB206. This confirms that the low affinity CD3 variable domain found in LB218 is less potent than that in LB206 in the primary T cell environment.

[0211] Next, selected LB proteins were evaluated for their ability to induce tumor cell killing by primary T cells in PD-L1+ high RKO cells. As before, cancer cells were seeded for approximately 24 hours prior to the addition of primary T cells from two different donors and selected intact or digested LB proteins at concentrations ranging from 0.01 nM to 10 nM as indicated. Representative results are shown in Figure 9C. Cancer cell killing was assessed using the Incucyte® Live-Cell Analysis system by measuring total annexin V region over time. In contrast to PD-L1+ low A549 cells, digested LB206 induced strong and sustained cell killing in RKO cells even at 0.1 nM, whereas no or low background was observed with 1 nM intact LB206 protein. Similarly, intact LB218 did not induce RKO cancer cell killing even at 10 nM (Figure 9D). As previously observed in A549 cells, strong Annexin V signals above background were detected in samples with 10 nM and 1 nM digested LB218, but the onset of cell killing at the 1 nM concentration was significantly earlier in RKO cells than in A549 cells (PD-L1 low), confirming that PD-L1 expression levels determine the ability of LB proteins to induce cell killing.

[0212] Next, we evaluated selected LB proteins for their ability to induce tumor cell killing by primary T cells in another PD-L1+ high cell line, TNBC MDA-MB-231 cells. As before, tumor cells were seeded and grown for 24 hours before addition of primary T cells and selected intact or digested (for the indicated times) LB proteins at concentrations ranging from 0.01 nM to 10 nM. Cancer cell killing was assessed using the Incucyte® Live-Cell Analysis system by measuring total annexin V region over time. Digested LB206 and LB213 induced robust tumor cell killing of MDA-MB-231 cells over time (Figures 9E-9F). As previously observed, LB206 showed the most robust tumor cell killing over time at 10-fold lower concentrations compared to LB213, suggesting activation after prolonged exposure to MDA-MB-231 cells.

[0213] Efficacy of lead molecules in humanized mouse models of triple-negative breast cancer (TNBC) Selected lead LB proteins were evaluated for their in vivo performance. MDA-MB-231 cancer cells were inoculated subcutaneously into myeloid cytokine boosted (hGM-CSF + hIL3 + hIL4 + FLT3L) CD34+ NCG mice. Tumors grew to a mean volume of 80 mm as measured by caliper. 3When tumors showed no progression, mice were treated intraperitoneally with either 4.5 mg / kg, 8.5 mg / kg, or 12 mg / kg LB protein, IgG isotype control, or atezolizumab, 8xQ3D, as indicated. Tumor volumes were monitored every 3-4 days by caliper measurement. Mean tumor volumes are shown in Figure 10. LB206 induced tumor regression (Figure 10A, Figure 10D), LB220 induced strong tumor growth inhibition (Figure 10B), and LB213 dose-dependent tumor growth inhibition (Figure 10C), whereas anti-PD-L1 atezolizumab showed no tumor growth control over isotype control. In the LB206 group, 5 / 8 animals achieved sustained remission over 40 days (tumor volume = 0 mm) (Figure 10D). No toxicity was observed during dosing, and all mice maintained their body weight throughout the experiment, i.e., there was no significant change compared to the IgG control (Figure 10E). These data support the hypothesis that the LB protein design promotes robust efficacy in a highly immune-resistant tumor environment, such as MDA-MB-231, which constitutively expresses high levels of PD-L1, without inducing systemic toxicity, a surprising finding. This model has been performed multiple times in previously published studies in which immune-engaging bispecific antibodies (including CD3-inducing bispecific antibodies) targeting surface-expressed markers found on MDA-MB-231 cells were administered at high levels (several milligrams / kg) with little to no therapeutic success (Del Bano et al., Front. Immunol. 2019, 10:1593; Liu et al., J ImmunoTherapy of Cancer 2021, 9:e003468; Kemper et al., Life Sci Alliance 2022, 5(11):e202201481).

[0214] Although the present invention has been described with its preferred specific embodiments, it should be understood that the foregoing description and the following examples are intended to be illustrative and not limiting of the scope of the invention. It will be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the scope of the invention, and furthermore, other aspects, advantages and modifications will be apparent to those skilled in the art to which the invention pertains. In addition to the embodiments described herein, the present invention contemplates and claims those inventions that result from combinations of the features of the inventions cited herein and the features of the cited prior art references that complement the features of the invention. Similarly, it is recognized that any described material, feature, or article may be used in combination with any other material, feature, or article, and such combinations are considered to be within the scope of the invention. The disclosures of each patent, patent application, and publication cited or described in this document are each incorporated herein by reference in their entirety for all purposes.

[0215] Numbered embodiments Notwithstanding the appended claims, the present disclosure includes the following numbered embodiments:

[0216] 1. A protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, and a second CH1 domain; the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL domain, and a second immunoglobulin light chain constant region; protein.

[0217] 2. A protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 light chain variable (VL) domain, and a first immunoglobulin light chain constant region; the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 VL domain, a second immunoglobulin light chain constant region, a second linker, an anti-CD3 VH domain, and a second CH1 domain; protein.

[0218] 3. The protein of embodiment 1 or 2, wherein the heavy chain comprises, in order from N-terminus to C-terminus, the anti-PD-L1 VH domain, the first CH1 domain, the first linker, the anti-CD3 VH domain, the second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0219] 4. A protein described in any one of embodiments 1 to 3, wherein the protein further comprises a third polypeptide chain comprising a hinge, a CH2 domain, and a CH3 domain.

[0220] 5. The protein of embodiment 4, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124.

[0221] 6. The protein of embodiment 1, wherein the protein further comprises a moiety that provides half-life extension.

[0222] 7. The protein of embodiment 6, wherein the moiety that provides half-life extension is polyethylene glycol (PEG) or an albumin binding domain.

[0223] 8. A protein comprising a first polypeptide chain comprising a heavy chain and a second polypeptide chain comprising a light chain, the heavy chain comprises, from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 VH domain, a second CH1 domain, a first hinge, a first CH2 domain, and a first CH3 domain; the light chain comprises, from N-terminus to C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 VL domain, a second immunoglobulin light chain constant region, a second hinge, a second CH2 domain, and a second CH3 domain; protein.

[0224] 9. The protein according to any one of embodiments 1 to 8, wherein the first linker comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 12.

[0225] 10. The protein according to any one of embodiments 1 to 9, wherein the second linker comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 12.

[0226] 11. the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 25; 11. The protein according to any one of embodiments 1 to 10.

[0227] 12. the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; 12. The protein according to any one of embodiments 1 to 11.

[0228] 13. the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; 12. The protein according to any one of embodiments 1 to 11.

[0229] 14. the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22; the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 25; the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; 11. The protein according to any one of embodiments 1 to 10.

[0230] 15. the anti-PD-L1 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 22;

[0231] the anti-PD-L1 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 25; the anti-CD3 VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; the anti-CD3 VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 30, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 32; 11. The protein according to any one of embodiments 1 to 10.

[0232] 16. The protein of any one of embodiments 1-15, wherein the anti-PD-L1 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 33, and 54-107.

[0233] 17. The protein of any one of embodiments 1 to 16, wherein the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 34.

[0234] 18. The protein of any one of embodiments 1-17, wherein the anti-PD-L1 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 33, and 54-107, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO: 34.

[0235] 19. The protein of any one of embodiments 1 to 17, wherein the anti-CD3 VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 44 to 48.

[0236] 20. The protein of any one of embodiments 1 to 19, wherein the anti-CD3 VL domain comprises the amino acid sequence of any one of SEQ ID NOs: 37 to 42.

[0237] 21. The protein of any one of embodiments 1 to 20, wherein the heavy chain comprises an amino acid sequence of any one of SEQ ID NOs: 116 to 123, 129 to 132, 137 to 141, 142, 144, 146, 147, 148, 150, and 152.

[0238] 22. The protein of any one of embodiments 1 to 21, wherein the light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 108-115, 125-128, 133-136, 143, 145, 151, and 153.

[0239] 23. (a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 122, the light chain comprises the amino acid sequence of SEQ ID NO: 114, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (b) the heavy chain comprises the amino acid sequence of SEQ ID NO: 123, the light chain comprises the amino acid sequence of SEQ ID NO: 115, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (c) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (d) the heavy chain comprises the amino acid sequence of SEQ ID NO: 117, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (e) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (f) the heavy chain comprises the amino acid sequence of SEQ ID NO: 119, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (g) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (h) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (j) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (k) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (l) the heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (m) said heavy chain comprises the amino acid sequence of SEQ ID NO: 120, said light chain comprises the amino acid sequence of SEQ ID NO: 111, and said third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (n) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 112, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (o) the heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 113, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (p) the heavy chain comprises the amino acid sequence of SEQ ID NO: 121, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (q) the heavy chain comprises the amino acid sequence of SEQ ID NO: 131, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; or (r) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; 5. The protein of embodiment 4.

[0240] 24. The protein of any one of embodiments 2 to 23, wherein the heavy chain comprises an IgG, IgE, IgM, IgD, IgA, or IgY constant region.

[0241] 25. The protein of any one of embodiments 2 to 23, wherein the heavy chain comprises an IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 constant region.

[0242] 26. A protein described in any one of embodiments 2 to 23, wherein the heavy chain comprises an immunologically inert constant region.

[0243] 27. The protein of any one of embodiments 2-23, wherein the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A and G237A, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, or a human IgG4 constant region comprising the amino acid substitution S228P, where numbering is according to the EU index as in Kabat.

[0244] 28. An immunoconjugate comprising a protein according to any one of embodiments 1 to 27 linked to a therapeutic agent.

[0245] 29. The immunoconjugate of embodiment 28, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0246] 30. A pharmaceutical composition comprising a protein according to any one of embodiments 1 to 27, or an immunoconjugate according to embodiment 28 or 29, and a pharma- ceutically acceptable carrier.

[0247] 31. A nucleic acid molecule encoding (a) the heavy chain amino acid sequence; (b) the light chain amino acid sequence; or (c) the amino acid sequences of both the heavy chain and the light chain of the protein described in any one of embodiments 1 to 27.

[0248] 32. An expression vector comprising the nucleic acid molecule of embodiment 31.

[0249] 33. A recombinant host cell comprising the nucleic acid molecule of embodiment 31 or the expression vector of embodiment 32.

[0250] 34. A method for producing a protein, the method comprising culturing a recombinant host cell described in embodiment 33 under conditions in which a nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0251] 35. A method for enhancing an anti-cancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein described in any one of embodiments 1 to 27, an immunoconjugate described in embodiment 28 or 29, or a pharmaceutical composition described in embodiment 30.

[0252] 36. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein described in any one of embodiments 1 to 27, an immunoconjugate described in embodiment 28 or 29, or a pharmaceutical composition described in embodiment 30.

[0253] 37. A method for ameliorating a symptom of cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein described in any one of embodiments 1 to 27, an immunoconjugate described in embodiment 28 or 29, or a pharmaceutical composition described in embodiment 30.

[0254] 38. The method of any one of embodiments 35 to 37, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or blood tissue cancer.

[0255] 39. A protein described in any one of embodiments 1 to 27, an immunoconjugate described in embodiment 28 or 29, or a pharmaceutical composition described in embodiment 30, for use in enhancing an anti-cancer immune response in a subject.

[0256] 40. A protein described in any one of embodiments 1 to 27, an immunoconjugate described in embodiment 28 or 29, or a pharmaceutical composition described in embodiment 30, for use in treating cancer in a subject.

[0257] 41. A protein described in any one of embodiments 1 to 27, an immunoconjugate described in embodiment 28 or 29, or a pharmaceutical composition described in embodiment 30, for use in ameliorating cancer symptoms in a subject.

[0258] 42. The protein, immunoconjugate or pharmaceutical composition for use according to any one of embodiments 39 to 42, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oral cavity or pharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or cancer of the blood tissue.

Claims

1. A protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, (a) The heavy chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 heavy chain variable (VH) domain, and a second CH1 domain, The light chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD3 light chain variable (VL) domain, and a second immunoglobulin light chain constant region; or (b) The heavy chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD3 light chain variable (VL) domain, and a first immunoglobulin light chain constant region. The light chain comprises, in order from the N-terminus to the C-terminus, an anti-PD-L1 light chain variable (VL) domain, a second immunoglobulin light chain constant region, a second linker, an anti-CD3 heavy chain variable (VH) domain, and a second CH1 domain. protein.

2. The protein according to claim 1, further comprising a third polypeptide chain having a first hinge, a first CH2 domain, and a first CH3 domain in order from the N-terminus to the C-terminus.

3. The protein according to claim 2, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

124.

4. The protein according to claim 1, wherein the first linker or the second linker each independently comprises an amino acid sequence having at least 95% sequence identity with respect to any one of the amino acid sequences of SEQ ID NOs: 1 to 12.

5. The protein according to claim 4, wherein the first linker or the second linker each independently comprises one amino acid sequence from SEQ ID NOs: 1 to 12.

6. The first linker and the second linker are (a) Sequence ID 1 and Sequence ID 2, respectively; (b) Sequence ID 3 and Sequence ID 4, respectively; (c) Sequence ID 3 and Sequence ID 6, respectively; (d) Sequence ID 1 and Sequence ID 5, respectively; (e) Sequence ID 6 and Sequence ID 4, respectively; (f) Sequence ID 5 and Sequence ID 5, respectively; (g) Sequence ID 6 and Sequence ID 6, respectively; (h) Sequence ID 6 and Sequence ID 3, respectively; (i) Sequence ID 6 and Sequence ID 1, respectively; (j) Sequence ID 1 and Sequence ID 4, respectively; (k) Sequence ID 3 and Sequence ID 2, respectively; or (l) Sequence ID 5 and Sequence ID 2, respectively. The protein according to claim 1, comprising the amino acid sequence.

7. The protein according to claim 1, wherein the anti-PD-L1 heavy chain variable (VH) domain comprises a heavy chain complementary region 1 (HCDR1) containing the amino acid sequence of SEQ ID NO: 20, a heavy chain complementary region 2 (HCDR2) containing the amino acid sequence of SEQ ID NO: 21, and a heavy chain complementary region 3 (HCDR3) containing the amino acid sequence of SEQ ID NO: 22; and the anti-PD-L1 light chain variable (VL) domain comprises a light chain complementary region 1 (LCDR1) containing the amino acid sequence of SEQ ID NO: 23, a light chain complementary region 2 (LCDR2) containing the amino acid sequence of SEQ ID NO: 24, and a light chain complementary region 3 (LCDR3) containing the amino acid sequence of SEQ ID NO:

25.

8. (a) The anti-CD3 heavy chain variable (VH) domain comprises HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, HCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 29; The anti-CD3 light chain variable (VL) domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 30, LCDR2 containing the amino acid sequence of SEQ ID NO: 31, and LCDR3 containing the amino acid sequence of SEQ ID NO: 32; or (b) The anti-CD3 heavy chain variable (VH) domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 26, HCDR2 containing the amino acid sequence of SEQ ID NO: 28, and HCDR3 containing the amino acid sequence of SEQ ID NO: 29; The anti-CD3 light chain variable (VL) domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 30, LCDR2 containing the amino acid sequence of SEQ ID NO: 31, and LCDR3 containing the amino acid sequence of SEQ ID NO:

32. The protein according to claim 1.

9. (a) The anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 20, HCDR2 containing the amino acid sequence of SEQ ID NO: 21, and HCDR3 containing the amino acid sequence of SEQ ID NO: 22; The anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 23, LCDR2 containing the amino acid sequence of SEQ ID NO: 24, and LCDR3 containing the amino acid sequence of SEQ ID NO: 25; The anti-CD3 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 26, HCDR2 containing the amino acid sequence of SEQ ID NO: 27, and HCDR3 containing the amino acid sequence of SEQ ID NO: 29; The anti-CD3 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 30, LCDR2 containing the amino acid sequence of SEQ ID NO: 31, and LCDR3 containing the amino acid sequence of SEQ ID NO: 32; or (b) The anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 20, HCDR2 containing the amino acid sequence of SEQ ID NO: 21, and HCDR3 containing the amino acid sequence of SEQ ID NO: 22; The anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 23, LCDR2 containing the amino acid sequence of SEQ ID NO: 24, and LCDR3 containing the amino acid sequence of SEQ ID NO: 25; The anti-CD3 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO: 26, HCDR2 containing the amino acid sequence of SEQ ID NO: 28, and HCDR3 containing the amino acid sequence of SEQ ID NO: 29; The anti-CD3 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO: 30, LCDR2 containing the amino acid sequence of SEQ ID NO: 31, and LCDR3 containing the amino acid sequence of SEQ ID NO:

32. The protein according to claim 1.

10. The protein according to claim 1, wherein the anti-PD-L1 heavy chain variable (VH) domain comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs. 33 and 54-107.

11. The protein according to claim 10, wherein the anti-PD-L1 VH domain comprises any one of the amino acid sequences of SEQ ID NO: 33 and 54-107.

12. The protein according to claim 1, wherein the anti-PD-L1 light chain variable (VL) domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

34.

13. The protein according to claim 12, wherein the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO:

34.

14. The protein according to claim 1, wherein the anti-CD3 heavy chain variable (VH) domain comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 44 to 48.

15. The protein according to claim 14, wherein the anti-CD3 VH domain comprises any one amino acid sequence of SEQ ID NOs: 44 to 48.

16. The protein according to claim 1, wherein the anti-CD3 light chain variable (VL) domain comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs. 37 to 42.

17. The protein according to claim 16, wherein the anti-CD3 VL domain comprises any one amino acid sequence of SEQ ID NOs. 37 to 42.

18. The protein according to claim 1, wherein the heavy chain comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs: 116-123, 129-132, 137-141, 142, 144, 146, 147, 148, 150, and 152.

19. The protein according to claim 18, wherein the heavy chain comprises the amino acid sequences of SEQ ID NOs: 116-123, 129-132, 137-141, 142, 144, 146, 147, 148, 150, and 152.

20. The protein according to claim 1, wherein the light chain comprises an amino acid sequence that is at least 90% identical to any one of the amino acid sequences of SEQ ID NOs. 53, 108-115, 125-128, 133-136, 143, 145, 151, and 153.

21. The protein according to claim 20, wherein the light chain comprises one of the amino acid sequences of SEQ ID NOs. 53, 108-115, 125-128, 133-136, 143, 145, 151, and 153.

22. (a) The heavy chain comprises the amino acid sequence of SEQ ID NO: 122, the light chain comprises the amino acid sequence of SEQ ID NO: 114, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (b) The heavy chain comprises the amino acid sequence of SEQ ID NO: 123, the light chain comprises the amino acid sequence of SEQ ID NO: 115, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (c) The heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (d) The heavy chain comprises the amino acid sequence of SEQ ID NO: 117, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (e) The heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (f) The heavy chain comprises the amino acid sequence of SEQ ID NO: 119, the light chain comprises the amino acid sequence of SEQ ID NO: 109, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (g) The heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (h) The heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (i) The heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (j) The heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 110, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (k) The heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (l) The heavy chain comprises the amino acid sequence of SEQ ID NO: 118, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (m) The heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 111, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (n) The heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 112, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (o) The heavy chain comprises the amino acid sequence of SEQ ID NO: 116, the light chain comprises the amino acid sequence of SEQ ID NO: 113, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (p) The heavy chain comprises the amino acid sequence of SEQ ID NO: 121, the light chain comprises the amino acid sequence of SEQ ID NO: 108, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (q) The heavy chain comprises the amino acid sequence of SEQ ID NO: 131, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; or (r) The heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the light chain comprises the amino acid sequence of SEQ ID NO: 127, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

124. The protein according to claim 2.

23. The protein according to claim 1, wherein the heavy chain includes a constant region of IgG, IgE, IgM, IgD, IgA, or IgY.

24. The protein according to claim 23, wherein the heavy chain comprises a constant region of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

25. The protein according to claim 1, wherein the heavy chain includes an immunologically inactive constant region.

26. The protein according to claim 23, wherein the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region containing amino acid substitutions L234A, L235A and G237A, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, or a human IgG4 constant region containing amino acid substitution S228P, wherein the numbering follows the EU index as found in Kabat.

27. ​​A pharmaceutical composition comprising the protein described in Claim 1 and a pharmaceutically acceptable carrier.

28. An immunoconjugate comprising the protein described in claim 1, linked to a therapeutic agent.

29. The immunoconjugate according to claim 28, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell proliferation inhibitory enzyme, a cell lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or an apoptosis-promoting agent.

30. The protein according to claim 1 (a) The heavy chain amino acid sequence; (b) The light chain amino acid sequence; or (c) The amino acid sequences of both the heavy chain and the light chain, A nucleic acid molecule that codes for something.

31. An expression vector comprising the nucleic acid molecule described in claim 30.

32. A recombinant host cell comprising the nucleic acid molecule described in claim 30 or an expression vector comprising the nucleic acid molecule described in claim 30.

33. A method for producing a protein, wherein the method is Culture the recombinant host cells described in claim 32 under conditions in which nucleic acid molecules are expressed, thereby producing the protein; and Isolating the protein from the host cells or culture, A method of including.

34. A composition comprising a protein according to any one of claims 1 to 26, an immunoconjugate according to claim 28 or 29, a nucleic acid molecule according to claim 30, an expression vector according to claim 31, or a recombinant host cell according to claim 32, or a pharmaceutical composition according to claim 27, for use in the treatment of cancer in a subject.

35. A composition comprising a protein according to any one of claims 1 to 26, an immunoconjugate according to claim 28 or 29, a nucleic acid molecule according to claim 30, an expression vector according to claim 31, or a recombinant host cell according to claim 32, or a pharmaceutical composition according to claim 27, for use in enhancing an anti-cancer immune response in a subject.

36. A composition or pharmaceutical composition for use in the treatment of cancer in a subject or in the enhancement of an anti-cancer immune response in a subject, comprising a protein according to any one of claims 1 to 26, an immune conjugate according to claim 28 or 29, a nucleic acid molecule according to claim 30, an expression vector according to claim 31, or a recombinant host cell according to claim 32, or according to claim 27, wherein the cancer is gastrointestinal stromal cancer (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, gastric cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, oropharyngeal cancer, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small intestine cancer, appendiceal cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, or hematological cancer.

Images