Activatable bispecific anti-CD28 and anti-PD-L1 proteins and uses thereof

Abstract

Provided herein are protein molecules that specifically bind to PD-L1 and also exhibit activatable specific CD28 binding in diseased tissue. Further provided herein are uses of such protein molecules for the treatment of cancer.

Description

Cross-references

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 497,084, filed April 19, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to activatable bispecific proteins and the treatment of cancer. Background Technology

[0003] In immuno-oncology therapy, a few key drug targets are expressed only in diseased tissues, while most are also expressed in non-disease-related tissues. Furthermore, many drugs used in cancer treatment employ potent cell-killing mechanisms. Therefore, drug binding to targets in non-disease-related tissues often leads to unwanted side effects.

[0004] PD-L1 is a cell surface receptor, a member of the immunoglobulin superfamily, and is primarily expressed on myeloid cells and regulatory T (Treg) cells in non-pathological tissues. However, PD-L1 has also been found to be highly expressed on some cancer cells. PD-L1 binds to the membrane protein PD1. The interaction between PD-L1 and PD1 on T cells downregulates T cell inflammatory activity, which promotes immune self-tolerance. PD-L1 is thus described as an immune checkpoint. Therefore, antagonistic anti-PD-L1 monoclonal antibodies that block the interaction with PD1 have shown the potential to act as well-tolerated immunotherapeutic agents in disease settings such as cancer by “liberating” anti-cancer T cell responses from the limitations of natural immunity. Thus, PD-L1 is a drug target for enhancing the anti-cancer effects of the adaptive immune system. However, in many cases, tumors may use various “escape mechanisms” to counteract the effects of anti-PD-L1 agents, one of which is the downregulation of co-stimulatory ligand molecules such as CD80 (B7.1) and CD86 (B7.2).

[0005] CD28 is the receptor for CD80 and CD86 proteins. It is a glycosylated, disulfide-linked 44 kDa homodimer expressed on the cell surface. Although CD28 is primarily expressed on T cells, it has also been found on other adaptive and innate immune cells. The binding and activation of CD28 by CD80 or CD86, particularly through binding to TCR signaling, provides a potent pro-immune signal that enhances T cell activation and memory.

[0006] Historically, antibodies targeting the extracellular domain of CD28 have been developed. Upon binding, these antibodies induce co-stimulatory activation signals in T lymphocytes, leading to increased inflammatory activity, proliferation, and cytolysis against infected or cancerous cells. Utilizing the binding domains from these antibodies, CD28-linked bispecific agents have also been developed, directing T cell co-stimulatory activity to target-enriched lesions. Therefore, anti-PD-L1 antibodies could be formulated into more potent and broader-spectrum therapeutics by acquiring the same CD28-binding ability, thereby strongly enhancing T cell killing mechanisms in anti-PD-L1 antibody-resistant disease environments. This would synergistically stimulate the function of key checkpoint inhibitors of the adaptive immune system and induceable “synthetic immunity.” However, the ability to make this combination effective in a single therapeutic agent structure (e.g., a standard bispecific antibody form with fully active PD-L1 and CD28 binding domains) is limited by the relatively broad expression profiles of both PD-L1 and CD28 across many cell types, such as T cells and myeloid cells. This broad expression profile may lead not only to dose-limiting toxicity of PD-L1 / CD28 binders but also to severe peripheral sedimentation / biodistribution problems, which limit the ability of such agents to achieve sufficiently high exposure in diseased tissues to utilize combinatorial mechanisms. Therefore, there is a need for engineered forms of bispecific binding proteins with activity that specifically targets the diseased tissue environment. Summary of the Invention

[0007] This article provides a protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, wherein the heavy chain comprises, in order from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD28 VH domain, and a second CH1 domain; wherein the light chain comprises, in order 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-CD28 VL domain, and a second immunoglobulin light chain constant region.

[0008] In some implementations, the heavy chain comprises, in order from N-end to C-end, an anti-PD-L1 VH domain, a first CH1 domain, a first connector, an anti-CD28 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0009] In some embodiments, the protein further comprises a third polypeptide chain including a hinge and an Fc region. In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:32.

[0010] In some embodiments, the first adapter comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the second adapter comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0011] In some embodiments, the anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:12, HCDR2 containing the amino acid sequence of SEQ ID NO:13, and HCDR3 containing the amino acid sequence of SEQ ID NO:14; the anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:15, LCDR2 containing the amino acid sequence of SEQ ID NO:16, and LCDR3 containing the amino acid sequence of SEQ ID NO:17; the anti-CD28 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:18, HCDR2 containing the amino acid sequence of SEQ ID NO:19, and HCDR3 containing the amino acid sequence of SEQ ID NO:20; and the anti-CD28 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:21, LCDR2 containing the amino acid sequence of SEQ ID NO:22, and LCDR3 containing the amino acid sequence of SEQ ID NO:23.

[0012] In some embodiments, the anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO:24, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO:25. In some embodiments, the anti-CD28 VH domain comprises the amino acid sequence of SEQ ID NO:26. In some embodiments, the anti-CD28 VL domain comprises the amino acid sequence of SEQ ID NO:27.

[0013] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:31. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.

[0014] This article provides a protein, in which (a) The heavy chain contains the amino acid sequence of SEQ ID NO:30, the light chain contains the amino acid sequence of SEQ ID NO:28, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO:32; or (b) The heavy chain contains the amino acid sequence of SEQ ID NO:31, the light chain contains the amino acid sequence of SEQ ID NO:29, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO:32.

[0015] In some embodiments, the heavy chain comprises constant regions of IgG, IgE, IgM, IgD, IgA, or IgY. In some embodiments, the heavy chain comprises constant regions of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the heavy chain comprises an immune-inert constant region. In some embodiments, the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region comprising amino acid substitutions for 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 an amino acid substitution for S228P, wherein the numbering is based on an EU index such as in Kabat.

[0016] This document provides an immunoconjugate comprising a disclosed protein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell-inhibiting enzyme, a cell-lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0017] This article provides a pharmaceutical composition comprising the protein or immunoconjugate disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0018] This article provides a nucleic acid molecule that encodes (a) the heavy chain amino acid sequence of the protein disclosed herein; (b) the light chain amino acid sequence; or (c) both the heavy chain amino acid sequence and the light chain amino acid sequence.

[0019] This article provides an expression vector containing the nucleic acid molecules disclosed herein.

[0020] This article provides a recombinant host cell containing the nucleic acid molecules or expression vectors disclosed herein.

[0021] This article provides a method for producing proteins, comprising: culturing a recombinant host cell containing an expression vector disclosed herein under conditions in which nucleic acid molecules can be expressed, thereby producing proteins; and isolating proteins from the host cell or culture.

[0022] This article provides a method for enhancing an anticancer 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.

[0023] This article provides a method 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.

[0024] This article provides a method for alleviating cancer symptoms in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0025] In some implementations of the methods provided herein, the cancer is gastrointestinal stromal carcinoma (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy. Attached Figure Description

[0026] Figure 1A This diagram illustrates exemplary asymmetric single-arm constructs of protein molecules disclosed herein, presenting their intact conformation (left), activated protease-cleaved conformation (middle), and inactivated protease-cleaved conformation (right). In the intact conformation, the anti-PD-L1 Fab binding domain is exposed and capable of binding its homologous target. Linkers in the heavy and light chains inhibit the binding of the anti-CD28 Fab domain; these linkers are both proteolytically cleaved and sequentially cleaved by matrix metalloproteinases (MMPs) and / or cathepsins. The first cleavage event produces an intermediate active state, enabling both anti-PD-L1 and anti-CD28 Fab from the single protein construct to bind their homologous targets, potentially guiding CD28+ T cell activation within the PD-L1+ immunosuppressive microenvironment. The second cleavage dissociates the anti-PD-L1 and anti-CD28 Fab, eliminating the ability of a single molecule to simultaneously bind both PD-L1 and CD28. Figure 1B The symmetrical two-arm and symmetrical one-arm constructs of the protein molecules described in this paper are depicted. Figure 1BTwo schematic diagrams are depicted. One diagram shows a complete symmetrical two-armed LB (LPD) protein (left) composed of two heavy chains and two light chains, and the other diagram shows a complete symmetrical one-armed LB (LPD) protein (right) composed of heavy and light chains. The symmetrical two-armed LPD protein consists of two light chain segments, each consisting of two Fab segments linked by a lower hinge joint (LHL). Each heavy chain consists of two Fab segments linked by a lower hinge joint (LHL), an Fc hinge, and an Fc segment containing both CH2 and CH3 domains (left). The symmetrical one-armed LB protein consists of a single light chain molecule composed of two Fab and Fc segments linked by a lower hinge joint (LHL), the Fc segment containing both a CH2 domain with an N-linked glycosylation site (CH2) or a Knobs mutation (CH2). The light chain is paired with a heavy chain, which consists of two Fab, Fc hinges and Fc fragments connected by a lower hinge joint (LHL), the Fc fragment containing both the CH2 and CH3 domains with an N-linked glycosylation site (CH2) or a Knobs mutation (CH3) (right).

[0027] Figure 2 A schematic diagram illustrating the activity mechanism of the activatable bispecific protein molecule presented in this paper is provided. Since the release activity of the CD28 binding domain is essentially monomeric, it cannot directly induce CD28 activation. Instead, it depends on binding to PD-L1+ cells, leading to the presentation of CD28 in a trans-multivalent form to nearby immune cells to induce CD28 signaling. The activated molecule thus simultaneously blocks PD-L1 / PD1 signaling to restore the uninhibited TCR "Signal 1" and provides CD28 signaling to amplify T cell activity via "Signal 2".

[0028] Figure 3 The surface plasmon resonance (SPR) combination of each CD28 V domain (in monovalent Fab form) with human recombinant CD28 was depicted.

[0029] Figure 4 This study demonstrates the ELISA binding of purified IgG_001 and IgG_002 or TGN1412 (IgG4) to human / cynomolgus monkey and mouse CD28 protein. The negative control protein used was the human IgG1 isotype. The binding levels of TGN1412, IgG_001, and IgG_002 to human / cynomolgus monkey CD28 were similar, and no signal was detected in the isotype control. No protein binding to the mouse CD28 recombinant protein was detected.

[0030] Figure 5 depicts MMP12-digested LPD proteins running on SDS-PAGE under reducing or non-reducing conditions. After incubation with MMP12 for a specified time, exemplary LPB proteins -1, -2, -3, -6, -7, -8, -9, -10, -11, and -12 were loaded onto reducing / non-reducing SDS-PAGE, respectively. Figure 5A The digestion profiles of LPD28-1 and LPD28-2 proteins after incubation with MMP12 for 0 h, 1 h, 2 h, 4 h, and 8 h are shown. Notably, prominent 25 kDa and 50 kDa bands appeared in the reducing / non-reducing SDS-PAGE, respectively, due to light chain cleavage at 75 kDa under reducing conditions (and 150 kDa under non-reducing conditions) and the disappearance of intact heavy chains. The simultaneous appearance of the lower band and the disappearance of the higher band indicates that the linkers in both the heavy and light chains were cleaved. Figure 5B The digestion profiles of LPD28-3 protein incubated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h are shown. Notably, prominent 25 kDa and 50 kDa bands appeared in the reducing / non-reducing SDS-PAGE, respectively, due to light chain cleavage at 75 kDa under reducing conditions (and 150 kDa under non-reducing conditions) and the disappearance of intact heavy chains. The simultaneous appearance of the lower band and the disappearance of the higher band indicates that the linkers in both the heavy and light chains were cleaved. Figure 5C The digestion spectra of LPB28-6 incubated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h are shown. Notably, prominent 25 kDa and 50 kDa bands appeared in the reduced SDS-PAGE, which is due to the cleavage of light and heavy chains at 75 kDa under reducing conditions (and 150 kDa under non-reducing conditions) and the accompanying loss of intact chains. Figure 5D The digestion spectra of LPB28-7 incubated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h are shown. Notably, prominent 25 kDa and 50 kDa bands appeared in the reduced SDS-PAGE, which is due to the cleavage of light and heavy chains at 75 kDa under reducing conditions (and 150 kDa under non-reducing conditions) and the accompanying loss of intact chains. Figure 5EThe digestion spectra of LPB28-8 and LPD28-9 incubated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h are shown. Notably, prominent 25 kDa and 50 kDa bands appeared in the reduced SDS-PAGE, which is due to the cleavage of light and heavy chains at 75 kDa under reducing conditions (and 150 kDa under non-reducing conditions) and the accompanying loss of intact chains. Figure 5F Digestion spectra of LPB28-10 incubated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h are shown. Notably, prominent bands of 25 kDa and approximately 55 kDa appeared in the reduced SDS-PAGE, and bands of approximately 50 kDa and approximately 120 kDa appeared in the reduced SDS-PAGE. This is due to the cleavage of the light chain at 75 kDa under reducing conditions (and at 250 kDa under non-reducing conditions) and the disappearance of the intact heavy chain. The simultaneous appearance of the lower bands and the disappearance of the higher bands indicates that the joints in both the heavy and light chains were cleaved. Figure 5G Digestion spectra of LPB28-11 incubated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h are shown. Notably, prominent 25 kDa and approximately 55 kDa bands appeared in the reduced SDS-PAGE, and approximately 50 kDa and approximately 120 kDa bands appeared in the reduced SDS-PAGE. This is due to light chain cleavage at 75 kDa under reducing conditions (and 250 kDa under non-reducing conditions) and the disappearance of intact heavy chains. The simultaneous appearance of the lower bands and the disappearance of the higher bands indicates that the joints in both the heavy and light chains were cleaved. Figure 5H Digestion spectra of LPB28-12 and LPD28-13 incubated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h are shown. Notably, prominent 25 kDa and approximately 55 kDa bands appeared in the reduced SDS-PAGE, while approximately 50 kDa and approximately 120 kDa bands appeared in the non-reduced SDS-PAGE. This is due to light chain cleavage at 75 kDa under reducing conditions (and 250 kDa under non-reducing conditions) and the disappearance of intact heavy chains. The simultaneous appearance of the lower bands and the disappearance of the higher bands indicates that the linkers in both heavy and light chains were cleaved. As shown in the box indicating lane movement, the loading error was corrected in the reducing gel.

[0031] Figure 6AThe ELISA results show the binding of purified IgG_001 and IgG_002 to human / cynomolgus monkey CD28 protein. Purified IgG_001 and IgG_002 proteins were used as positive controls, while human IgG1 isotype was used as a negative control. Figure 6B The binding of recombinant human / cynomolgus monkey CD28 protein to intact LPD28-3 or MMP12-treated LPD28-3 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h was demonstrated. Intact LPD28-3 showed binding only at the highest concentration tested, while all MMP12-treated LPD28-3 proteins showed strong binding at all tested concentrations. Figure 6C The binding of recombinant human / cynomolgus monkey CD28 protein to intact LPD28-10 or MMP12-treated LPD28-10 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h was demonstrated. Intact LPD28-10 showed binding only at the highest concentration tested, while all MMP12-treated LPD28-10 proteins showed strong binding at all tested concentrations. Figure 6D The binding of recombinant human / cynomolgus monkey CD28 protein to intact LPD28-11 or LPD28-11 treated with MMP12 at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h was shown. Intact LPD28-11 showed some level of binding only at the highest concentration tested, while all MMP12-treated LPD28-11 proteins showed strong binding.

[0032] Figure 7A The binding of Jurkat cells to intact LPD28-3 or LPD28-3 treated with MMP12 at 0 h, 15 min, 1 h, and 8 h was depicted. No binding of Jurkat cells to intact LPD28-3 (0 min) or the isotype control was observed. In contrast, all MMP12-treated samples showed similar binding levels to Jurkat cells. Figure 7B The binding of Jurkat cells to intact LPD28-10 or LPD28-10 treated with MMP12 at 0 h, 15 min, 1 h, and 8 h was depicted. No binding of Jurkat cells to intact LPD28-10 (0 min) or the isotype control was observed. In contrast, all MMP12-treated samples showed similar binding levels to Jurkat cells. Figure 7CThe binding of Jurkat cells to intact LPD28-11 or LPD28-11 treated with MMP12 at 0 h, 15 min, 1 h, and 24 h was depicted. Low levels of binding of Jurkat cells to intact LPD28-11 (0 min) were observed. No binding was detected with the isotype control. In contrast, all MMP12-treated samples showed similar binding levels to Jurkat cells.

[0033] Figure 8 The binding of CD28 to IgG protein and its activity on Jurkat IL2 reporter cells with and without CD3 (SP34) stimulation were depicted. In the absence of CD3 (SP34) co-stimulation, luminescent signals of IgG_001 and TGN1412 were detected, but those of IgG_002 were not. In the presence of CD3 stimulation, both TGN1412 and IgG_002 showed significantly increased luminescent signals compared to CD3 stimulation alone (as shown by the dashed lines).

[0034] Figure 9 depicts the activity of intact LPD protein or MMP12-treated LPD protein in the presence or absence of CD3 stimulation and in the presence or absence of human PDL1+MDA-MB-231 cells. Figure 9A Activity assays were performed on intact LPD28-1 and LPD28-2 and LPD28-2 treated with MMP12 for 2 h in the presence of CD3 stimulation and MDA-MB-231 cells. Intact LPD28-1 showed no activity compared to CD3 stimulation levels, while LPD28-1 treated with MMP12 for 2 h showed a significant luminescent signal stimulation exceeding the CD3 signal. In contrast, LPD28-2 treated with MMP12 for 2 h showed no activity compared to CD3 stimulation levels. Figure 9B The activity assays of intact LPD28-3 and 2 h MMP12-treated LPD28-3 were shown in the presence of CD3 stimulation and MDA-MB-231 cells. Neither intact LPD28-3 nor MMP12-treated LPD28-3 showed activity compared to CD3 stimulation levels. Figure 9C The activity assays of intact LPD28-3 and 2-h MMP12-treated LPD28-3 were shown in the absence of CD3 stimulation and in the presence of MDA-MB-231 cells. Neither intact LPD28-3 nor MMP12-treated LPD28-3 showed activity compared to background levels. Figure 9DThe activity assays showed that intact LPD28-3 and 2-h MMP12-treated LPD28-3 were tested in the presence of CD3 stimulation but in the absence of MDA-MB-231 cells. Neither intact LPD28-3 nor MMP12-treated LPD28-3 showed any activity compared to the CD3 stimulation level. Figure 9E The activity assays of intact LPD28-3 and 2-h MMP12-treated LPD28-3 were shown in the absence of CD3 stimulation and MDA-MB-231 cells. Neither intact LPD28-3 nor MMP12-treated LPD28-3 showed activity compared to the background signal. Figure 9F The activity assays of intact LPD28-8 and MMP12-treated LPD28-8 were shown in the presence of CD3 stimulation and MDA-MB-231 cells. Neither intact LPD28-3 nor MMP12-treated LPD28-3 showed activity compared to CD3 stimulation levels. Figure 9G The activity assays of intact LPD28-8 and MMP12-treated LPD28-8 were shown in the absence of CD3 stimulation but in the presence of MDA-MB-231 cells. Neither intact LPD28-8 nor MMP12-treated LPD28-8 showed activity compared to background levels. Figure 9H The activity assays of intact LPD28-8 and MMP12-treated LPD28-8 were shown in the presence of CD3 stimulation but in the absence of MDA-MB-231 cells. Neither intact LPD28-8 nor MMP12-treated LPD28-8 showed activity compared to the CD3 stimulation level. Figure 9I The activity assays of intact LPD28-8 and MMP12-treated LPD28-8 were shown in the absence of CD3 stimulation and MDA-MB-231 cells. Neither intact LPD28-8 nor MMP12-treated LPD28-8 showed activity compared to the background signal. Figure 9J The activity assays of intact LPD28-9 and MMP12-treated LPD28-9 were shown in the presence of CD3 stimulation and MDA-MB-231 cells. Neither intact LPD28-9 nor MMP12-treated LPD28-9 showed activity compared to CD3 stimulation levels. Figure 9K The activity assays of intact LPD28-9 and MMP12-treated LPD28-9 were shown in the absence of CD3 stimulation and in the presence of MDA-MB-231 cells. Neither intact LPD28-9 nor MMP12-treated LPD28-9 showed activity compared to background levels. Figure 9LThe activity assays of intact LPD28-9 and MMP12-treated LPD28-9 were shown in the presence of CD3 stimulation but in the absence of MDA-MB-231 cells. Neither intact LPD28-9 nor MMP12-treated LPD28-9 showed activity compared to the CD3 stimulation level. Figure 9M The activity assays of intact LPD28-9 and MMP12-treated LPD28-9 were shown in the absence of CD3 stimulation and MDA-MB-231 cells. Neither intact LPD28-9 nor MMP12-treated LPD28-9 showed activity compared to the background signal.

[0035] Figure 10A The activity assays of intact LPD28-10 and MMP12-treated LPD28-10 were shown in the presence of CD3 stimulation and MDA-MB-231 cells. Intact LPD28-10 showed no activity compared to CD3 stimulation levels. All MMP12-treated samples showed significant luminescence induction compared to background signal. Figure 10B The activity assays of intact LPD28-10 and MMP12-treated LPD28-10 were shown in the absence of CD3 stimulation and in the presence of MDA-MB-231 cells. Intact LPD28-10 showed no activity compared to background levels. All MMP12-treated samples showed significant luminescence induction compared to background signals. Figure 10C The activity assay of intact LPD28-10 was shown in the presence of CD3 stimulation but in the absence of MDA-MB-231 cells. Intact LPD28-10 showed no activity compared to CD3 stimulation levels. Figure 10D The activity assays of intact LPD28-10 and MMP12-treated LPD28-10 were shown in the absence of CD3 stimulation and MDA-MB-231 cells. In the absence of CD3 stimulation and MDA-MB-231 cells, intact LPD28-10 showed no activity compared to the background signal, while all MMP12-treated samples showed significant activation compared to the background. Figure 10E The activity assays of intact LPD28-11 and MMP12-treated LPD28-11 were shown in the presence of CD3 stimulation and MDA-MB-231 cells. Intact LPD28-11 showed very low activity at the highest concentration compared to CD3 stimulation levels. All MMP12-treated samples showed significant luminescence induction compared to background signal. Figure 10FThe activity assays of intact LPD28-11 and MMP12-treated LPD28-11 were shown in the absence of CD3 stimulation and in the presence of MDA-MB-231 cells. Neither intact LPD28-11 nor MMP12-treated LPD28-11 showed activity compared to background levels. Figure 10G The activity assay of intact LPD28-11 was shown in the presence of CD3 stimulation but in the absence of MDA-MB-231 cells. Intact LPD28-11 showed lower activity levels at the highest concentration tested compared to CD3 stimulation levels. All MMP12-treated LPD28-11 samples showed activity. Figure 10H The activity assays of intact LPD28-11 and MMP12-treated LPD28-11 were shown in the absence of CD3 stimulation and MDA-MB-231 cells. Neither intact LPD28-11 nor MMP12-treated LPD28-11 showed activity compared to the background signal. Figure 10I The activity assays of intact LPD28-12 and MMP12-treated LPD28-12 were shown in the presence of CD3 stimulation and MDA-MB-231 cells. Intact LPD28-12 showed no activity compared to CD3 stimulation levels. All MMP12-treated samples showed significant luminescence induction compared to background signal. Figure 10J The activity assays of intact LPD28-12 and MMP12-treated LPD28-12 were shown in the absence of CD3 stimulation and in the presence of MDA-MB-231 cells. Intact LPD28-12 showed no activity compared to background levels. All MMP12-treated samples showed significant luminescence induction compared to background signals. Figure 10K The activity assay of intact LPD28-12 was demonstrated in the presence of CD3 stimulation but in the absence of MDA-MB-231 cells. Intact LPD28-12 showed some activity at the highest concentration tested compared to CD3 stimulation levels. All MMP12-treated samples showed significant luminescence induction compared to background signal. Figure 10L The activity assays of intact LPD28-12 and MMP12-treated LPD28-12 were shown in the absence of CD3 stimulation and MDA-MB-231 cells. In the absence of CD3 stimulation and MDA-MB-231 cells, intact LPD28-12 showed no activity compared to the background signal, while all MMP12-treated samples showed significant activation compared to the background. Figure 10MThe activity assays of intact LPD28-13 and MMP12-treated LPD28-13 were shown in the presence of CD3 stimulation and MDA-MB-231 cells. Intact LPD28-13 showed no activity compared to CD3 stimulation levels. All MMP12-treated samples showed significant luminescence induction compared to background signal. Figure 10N The activity assays of intact LPD28-13 and MMP12-treated LPD28-13 were shown in the absence of CD3 stimulation and in the presence of MDA-MB-231 cells. Intact LPD28-13 showed no activity compared to background levels. All MMP12-treated samples showed significant luminescence induction compared to background signals. Figure 10O The activity assay of intact LPD28-13 was shown in the presence of CD3 stimulation but in the absence of MDA-MB-231 cells. Intact LPD28-13 showed some activity compared to CD3 stimulation levels. All MMP12-treated samples showed significant luminescence induction compared to background signal. Figure 10P The activity assays of intact LPD28-13 and MMP12-treated LPD28-13 were shown in the absence of CD3 stimulation and MDA-MB-231 cells. In the absence of CD3 stimulation and MMP12-treated cells, intact LPD28-13 showed no activity compared to the background signal, while all MMP12-treated samples showed significant activation compared to the background.

[0036] Figure 11 shows the activity assay of the test sample in mixed lymphocyte reactions and in the presence or absence of human PDL1+MBA-MB-231 cells. Results for LPD28-10 from three different donor pairs are shown. Figure 11A shows IL2 release in the supernatant of the MLR assay in the absence (left) or presence (right) of MDA-MB-231 cells. Regardless of the test conditions, neither intact LPD28-1 protein nor MMP12-treated LPD28-1 protein resulted in an increase in IL2 release compared to background. Figure 11BThe figures show IFNg release in the supernatant of MLR assays in the absence (left) and presence (right) of MDA-MB-231 cells. Regardless of the testing conditions, neither intact LPD28-1 protein nor MMP12-treated LPD28-1 protein resulted in an increase in IFNg release compared to background. Figure 11C shows IL2 release in the supernatant of MLR assays in the absence of MDA-MB-231 cells. Intact LPD28-10 protein did not result in a detectable increase in IL2 release compared to PDL1 blockade (atezolizumab) alone. In contrast, MMP12-treated LPD28-10 resulted in a significant increase in IL2 release compared to background in the absence of PDL1 binding. Figure 11D shows IL2 release in the supernatant of MLR assays in the presence of MDA-MB-231 cells. Intact LPD28-10 protein did not result in a detectable increase in IL2 release compared to PDL1 blockade (atezolizumab) alone. In contrast, MMP12 treatment of LPD28-10 resulted in a significant increase in IL2 release compared to background. Figure 11E shows IFNg release in the supernatant of MLR measurements in the absence of MDA-MB-231 cells. Intact LPD28-10 protein resulted in a detectable increase in IFNg release compared to PDL1 blockade (atezolizumab) treatment alone. In contrast, in the absence of PDL1 conjugation, MMP12 treatment of LPD28-10 resulted in a significant increase in IFNg release compared to background. Figure 11F shows IFNg release in the supernatant of MLR measurements in the presence of MDA-MB-231 cells. Intact LPD28-10 protein did not result in a detectable increase in IFNg release compared to PDL1 blockade (atezolizumab) treatment alone. In contrast, MMP12 treatment of LPD28-10 resulted in a significant increase in IFNg release compared to background.

[0037] Figure 12 The purified IgG molecules TGN1412 or IgG_001 mediated by primary T cell killing in the cancer cell line (MDA-MB-231) were described using the Incucyte live cell analysis platform, along with the indicative conditions.

[0038] Figure 13 depicts the process via Incucyte ® The live cell analysis platform measured LPD protein, whether intact or incubated with MMP12, at indicated concentrations, to induce primary T cell-mediated cell killing in the cancer cell line MDA-MB-231. Figure 13A Primary T-cell killing mediated by LPD28-1 incubated with intact or 5 min MMP12-mediated cancer cell line MDA-MB-231, as measured by the Incucyte® live-cell analysis platform, was depicted. A positive control was a PDL1xCD3 conjugate used for comparison. Figure 13BPrimary T-cell killing mediated by LPD28-10 cells incubated with intact or 15- or 30-minute MMP12 was characterized using the Incucyte® live-cell analysis platform. Figure 13C Primary T-cell killing of cancer cell line MDA-MB-231 cells mediated by intact or 15 min MMP12 incubation was depicted using the Incucyte® live cell analysis platform. Figure 13D Primary T-cell killing of cancer cell line MDA-MB-231 cells mediated by intact or 15 min MMP12-incubated LPD28-13 cells, as measured by the Incucyte® live-cell analysis platform, was depicted.

[0039] Figure 14 depicts cytokine release data from PBMCs isolated from 20 healthy donors and incubated with intact LPD molecules or control proteins coated on 96-well plates. Incubation was stopped after 72 h, and the supernatant was analyzed for cytokine release as instructed. Figure 14A The release of IL2 was depicted by stimulation of positive and negative control proteins, buffer (PBS), isotype control, and intact LPD molecules. Figure 14B The release of IFNg stimulated by positive and negative control proteins, buffer (PBS), isotype control, and intact LPD molecules was depicted. Figure 14C The release of IL-10 was depicted by stimulation with positive and negative control proteins, buffer (PBS), isotype control, and intact LPD molecules. Figure 14D The release of IL-13 stimulated by positive and negative control proteins, buffer (PBS), isotype control, and intact LPD molecules was depicted. Figure 14E The release of TNFα stimulated by positive and negative control proteins, buffer (PBS), isotype control, and intact LPD molecules was depicted. Figure 14F The release of IL-6 was depicted by stimulation with positive and negative control proteins, buffer (PBS), isotype control, and intact LPD molecules.

[0040] Figure 15 depicts cytokine release data from PBMCs isolated from 10 healthy donors and incubated with intact LPD molecules or control proteins. LPD28-1 and LPD28-2 were incubated at three concentrations (C1 = 15 μg / mL, C2 = 2 μg / mL, and C3 = 1 μg / mL). Relevant negative and positive controls were included in the assays, such as blank / culture medium (negative control), LPS at one concentration (1 μg / mL), a commercially available CD28 superagonist at two concentrations (15 and 1 μg / mL), and Tegenero IgG1 and IgG4 at two concentrations (15 and 1 μg / mL). After 24 h, the supernatant was harvested and the secretion of IL-2, IL-6, IL-8, IL-10, IL-13, IFN-γ, and TNF-α was analyzed by multiplex analysis. Figure 15A The fold change in cytokine release induced by the indicated control protein or the intact LPD protein compared to the buffer control was depicted. Figure 15B The fold change in cytokine release induced by the indicated control protein or the intact LPD protein in the RESTORE assay compared to the buffer control was depicted.

[0041] Figure 16 The structure of an exemplary symmetrical two-arm construct of the protein molecule described herein is depicted. Detailed Implementation

[0042] Two key issues limit the efficacy of CD28-activating, tumor-targeting bispecific antibody drugs in oncology: 1) Antibody targets found on cancer cells (such as PD-L1) may be expressed in many different cell classes in vivo, not just on tumor cells. This extratumor target expression often leads to dose-limiting side effects because the CD28-binding domain in standard bispecific molecules is constitutively active and can therefore direct T cell activation in the presence of any target-positive cells, whether or not they are in diseased tissue. Extratumor target expression may also lead to an antigen “settling” effect, reducing the amount of drug that penetrates the tumor.

[0043] 2) CD28 positive cells are present in the bloodstream and have a high concentration in secondary lymphoid tissues, which produces a huge sedimentation effect on this arm, affecting biodistribution and the availability of free drugs for tumor penetration.

[0044] Both of the above factors minimize the potential safety and efficacy of bispecific antibodies driving CD28 binding and activation. The anti-PD-L1 and anti-CD28 proteins presented in this paper (see, e.g., Figure 1) overcome peripheral sedimentation and toxicity issues by minimizing CD28 binding outside of lesion tissue. This effect is achieved by adding a PD-L1 binding domain and a linker above the CD28 binding domain (i.e., the amino terminus). Using an appropriate combination of the upper domain and linker produces a conformation that minimizes binding activity in the lower (i.e., carboxyl terminus) CD28 domain. The PD-L1 domain then drives high concentrations in the PD-L1-rich tumor microenvironment. The protein construct linker system utilizes the elevated MMP and cathepsin activity commonly found in solid tumors to cleave the linker peptide, exposing the CD28 binding domain, thereby conditionally activating CD28 activation activity in the tumor rather than the periphery. The biological functions of these combinations thus give the molecules the potential to avoid peripheral CD28 sedimentation and maximize T-cell immune responses against cancer cells, such as… Figure 2 The overview.

[0045] This document discloses a protein with conditional activity in diseased human tissues. The disclosed protein exhibits full activity in specifically binding to and blocking systemic PD-L1; demonstrates minimized CD28 binding in healthy tissues; and becomes highly activated in CD28 binding and activation once in a PD-L1-positive diseased tissue environment. The disclosed protein contains a CD28-binding domain that is masked by a PD-L1-binding domain in non-disease-prone tissues. The protein also contains two peptide linkers that are cleaved by one or more proteases expressed in diseased tissues (e.g., tumors). Linker cleavage exposes the CD28-binding domain in diseased tissues, thus allowing the protein to selectively bind and / or function in diseased tissues.

[0046] protein molecules This article presents a protein comprising two Fab fragments (anti-PD-L1 Fab and anti-CD28 Fab). The protein is monovalent in its intact structure and, upon activation, can only have maximum monovalent CD28 binding to minimize the risk of peripheral toxicity associated with bivalent activated anti-CD28 antibodies.

[0047] In some embodiments, the protein comprises a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, wherein the heavy chain comprises, in N-terminal to C-terminal order, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD28 VH domain, and a second CH1 domain; wherein the light chain comprises, in N-terminal to C-terminal order, an anti-PD-L1 light chain variable (VL) domain, a first immunoglobulin light chain constant region, a second linker, an anti-CD28 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, in N-terminal to C-terminal order, an anti-PD-L1 VH domain, a first CH1 domain, a first linker, an anti-CD28 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0048] In some embodiments, the protein further comprises a third polypeptide chain containing a hinge and an Fc region. The third polypeptide chain may be referred to as an "Fc-residue".

[0049] Figure 1 shows a schematic diagram of an exemplary protein with labeled domains according to the present disclosure.

[0050] In some embodiments, the linkers described herein can be cleaved by proteases. In some embodiments, the proteases are present in diseased tissue. In some embodiments, the first and / or second linkers can be cleaved by matrix metalloproteinases (MMPs) and / or cathepsins present in diseased tissue, such as tumors. Linkers in proteins are immunoglobulin-derived hinge sequences that are both sensitive to proteolysis and can be sequentially cleaved, wherein the first cleavage takes the intact structure and produces an intermediate active state, enabling the anti-PD-L1 Fab and anti-CD28 Fab from a single protein construct to bind to their homologous targets. A second cleavage event in the second linker removes the covalent bond between the anti-PD-L1 Fab and anti-CD28 Fab, eliminating the molecule's ability to recruit T cells to kill PD-L1+ cells. Thus, this secondary cleavage event constitutes a "self-destruct mechanism" that minimizes the risk of the activating molecule escaping the tumor microenvironment. Cleavage linkers based on immunoglobulin hinge sequences can also recruit enhanced immune effector functions (antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent phagocytosis (ADCP)) at the cell membrane via endogenous anti-hinge antibodies, a phenomenon known in human patients with (or without) underlying autoreactive diseases.

[0051] In some implementations, the protein comprises one or more amino acid sequences provided in Table 1 or Table 2.

[0052] Table 1: Exemplary Sequences

[0053]

[0054]

[0055]

[0056]

[0057] In the VH and VL domain sequences, the CDR sequence is underlined.

[0058] Table 2 describes selected exemplary proteins described herein, generated using selected sequences as shown in Table 1. Figure 3 As shown, the two anti-CD28 Fab proteins were further characterized in the SPR binding assay.

[0059] Table 2: Exemplary strand sequence combinations of anti-CD3 Fab sequences

[0060] Table 3 describes selected IgG proteins generated using selected sequences as described in Table 1. Figure 4 and Figure 8 The two CD28 IgG proteins were further characterized in the ELISA binding assay and Jurkat reporter gene assay.

[0061] Table 3: Exemplary anti-CD3 IgG sequence combinations

[0062] Table 4: Full-length sequences of exemplary heavy and light chains

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] Anti-PD-L1 / anti-CD28 protein designs can be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and may or may not have effector functionality.

[0070] The proteins disclosed herein contain domains and regions of antibody molecules. The term "antibody" broadly refers to immunoglobulin (Ig) molecules, typically comprising 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 forms are known in the art.

[0071] In a full-length antibody, each heavy chain contains a heavy chain variable domain (abbreviated as VH domain) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. The constant regions of IgG, IgA, and IgD contain a flexible hinge region between the CH1 and CH2 domains. Each light chain contains a light chain variable domain (abbreviated as VL domain) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL domains can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and interspersed with more conserved regions called framework regions (FRs). Each VH and VL domain consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Frame regions and CDRs have been defined and described, for example, in Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; or IMGT Lefranc MP. Unique database numbering system for immunogenetic analysis. Immunol Today. Nov. 1997; 18(11):509.

[0072] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region can be a native sequence Fc region or a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226 or from Pro230 to its C-terminus. Residues in the Fc region are numbered according to the EU index, as in Kabat. The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH3. The Fc region can exist in dimer or monomeric form. The Fc region binds to various cellular receptors (such as Fc receptors) and other immune molecules (such as complement proteins).

[0073] The protein described in this article contains two Fab fragments. The Fab fragment is a monovalent antigen-binding fragment composed of the VL, VH, CL, and CH1 domains.

[0074] 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 typically contain two identical heavy chains and two identical light chains, as well as two antigen-binding domains, each consisting of a VH and a VL. Typically, IgA antibodies consist of two monomers, each consisting of two heavy chains and two light chains (as with IgG, IgD, and IgE antibodies); thus, the IgA molecule has four antigen-binding domains, each consisting of a VH and a VL. Some IgA antibodies are monomeric because they consist of two heavy chains and two light chains. Secretory IgM antibodies typically consist of five monomers, each consisting of two heavy chains and two light chains (as with IgG and IgE antibodies). Therefore, the IgM molecule has ten antigen-binding domains, each consisting 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. As used herein, the terms "immunobinding" and "immunobinding property" refer to a type of non-covalent interaction occurring between an immunoglobulin molecule (e.g., an antibody or its antigen-binding moiety) or a protein containing an immunoglobulin-derived binding domain and an antigen specific to that immunoglobulin or protein. The strength or affinity of an immunobinding interaction can be expressed by the dissociation constant (K0) of the interaction. d Let K be the smaller one. d This represents a higher affinity. The immunobinding properties of a selected peptide can be quantified using methods well-known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that also affect the rates in both directions. Therefore, the "association rate constant" (Kassociation rate constant) is used. on) and "dissociation rate constant" (K off Both can be determined by calculating the concentration and the actual association and dissociation rates. (See Malmqvist, Nature 361:186-187 (1993)). K off / K on The ratio can cancel out all parameters unrelated to affinity and is equal to the dissociation constant K. d (See Davies et al. (1990)) Annual Rev Biochem 59: 439-473). When the equilibrium binding constant (K) d When the concentration is ≤10 μM, preferably ≤10 nM, more preferably ≤10 nM, and most preferably ≤100 pM to about 1 pM, the antibody or antigen-binding moiety provided herein is considered to specifically bind to PD-L1 or CD28, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. A K-type assay for determining antibodies... d The method involves using surface plasmon resonance (SPR), typically employed with biosensor systems such as the Biacore® system.

[0075] Functionally, the protein binding affinity presented in this article can reach 10. -5 M to 10 -12 Within a certain range. For example, the binding affinity of the proteins provided in this article is from 10... -6 M to 10 -12 M, from 10 -7 M to 10 -12 M, from 10 -8 M to 10 -12 M, from 10 -9 M to 10 -12 M, from 10 -5 M to 10 -11 M, from 10 -6 M to 10 -11 M, from 10 -7 M to 10 -11 M, from 10 -8 M to 10 -11 M, from 10 -9 M to 10 -11 M, from 10 -10 M to 10 -11 M, from 10 -5 M to 10 -10 M, from 10 -6 M to 10 -10 M, from 10 -7 M to 10 -10M, from 10 -8 M to 10 -10 M, from 10 -9 M to 10 -10 M, from 10 -5 M to 10 -9 M, from 10 -6 M to 10 -9 M, from 10 -7 M to 10 -9 M, from 10 -8 M to 10 -9 M, from 10 -5 M to 10 -8 M, from 10 -6 M to 10 -8 M, from 10 -7 M to 10 -8 M, from 10 -5 M to 10 -7 M, from 10 -6 M to 10 -7 M or from 10 -5 M to 10 -6 M.

[0076] This article provides a protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, wherein the heavy chain comprises, in order from N-terminus to C-terminus, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first linker, an anti-CD28 VH domain, and a second CH1 domain; wherein the light chain comprises, in order 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-CD28 VL domain, and a second immunoglobulin light chain constant region.

[0077] In some implementations, the heavy chain comprises, in order from N-end to C-end, an anti-PD-L1 VH domain, a first CH1 domain, a first connector, an anti-CD28 VH domain, a second CH1 domain, a hinge, a CH2 domain, and a CH3 domain.

[0078] In some embodiments, the protein further comprises a third polypeptide chain including a hinge and an Fc region. In some embodiments, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:32.

[0079] In some embodiments, the first adapter comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4. In some embodiments, the second adapter comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0080] In some embodiments, the anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:12, HCDR2 containing the amino acid sequence of SEQ ID NO:13, and HCDR3 containing the amino acid sequence of SEQ ID NO:14; the anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:15, LCDR2 containing the amino acid sequence of SEQ ID NO:16, and LCDR3 containing the amino acid sequence of SEQ ID NO:17; the anti-CD28 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:18, HCDR2 containing the amino acid sequence of SEQ ID NO:19, and HCDR3 containing the amino acid sequence of SEQ ID NO:20; and the anti-CD28 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:21, LCDR2 containing the amino acid sequence of SEQ ID NO:22, and LCDR3 containing the amino acid sequence of SEQ ID NO:23.

[0081] In some embodiments, the anti-PD-L1 VH domain contains the amino acid sequence of SEQ ID NO:24, and the anti-PD-L1 VL domain contains the amino acid sequence of SEQ ID NO:25.

[0082] In some implementations, the anti-CD28 VH domain contains the amino acid sequence of SEQ ID NO:26.

[0083] In some implementations, the antiCD28 VL domain contains the amino acid sequence of SEQ ID NO:27.

[0084] In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:31. In some embodiments, the heavy chain comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:31.

[0085] In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29. In some embodiments, the heavy chain comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.

[0086] In some implementations, this document provides a protein, wherein (a) The heavy chain contains the amino acid sequence of SEQ ID NO:30, the light chain contains the amino acid sequence of SEQ ID NO:28, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO:32; or (b) The heavy chain contains the amino acid sequence of SEQ ID NO:31, the light chain contains the amino acid sequence of SEQ ID NO:29, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO:32.

[0087] This document also provides a protein comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence provided herein, having one, two, or three conserved amino acid substitutions; and wherein the light chain comprises the amino acid sequence provided herein, having one, two, or three conserved amino acid substitutions. In some embodiments, the conserved amino acid substitutions occur only in the FR sequence and not in the CDR sequence. In some embodiments, no conserved amino acid substitutions occur in the first or second adapter sequence.

[0088] In some embodiments, the protein provided herein comprises an immunoglobulin heavy chain constant region at the C-terminus of the heavy 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 immune-inert. In some embodiments, the immunoglobulin heavy chain constant region contains one or more mutations to reduce or prevent FcγR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the immunoglobulin heavy chain constant region is the wild-type human IgG1 constant region, the wild-type human IgG2 constant region, the wild-type human IgG4 constant region, the human IgG1 constant region containing amino acid substitutions L234A, L235A, and G237A, the human IgG1 constant region containing amino acid substitutions L234A, L235A, G237A, and P331S, or the human IgG4 constant region containing the amino acid substitution S228P, wherein the numbering is based on the EU index as in Kabat. In some embodiments, the positions of amino acid residues in the immunoglobulin molecule constant region are numbered according to the EU index as in Kabat (Ward et al., 1995). Therap. Immunol. 2: 77-94).

[0089] In some embodiments, the protein provided herein may comprise an immunoglobulin light chain constant region as a κ light chain. In some embodiments, the κ light chain comprises SEQ ID NO:7.

[0090] In some implementations, the protein provided herein may comprise an immunoglobulin light chain constant region as a λ light chain.

[0091] In some embodiments, the protein provided herein may comprise an immunoglobulin heavy chain constant region comprising the amino acid sequence of the Fc region of human IgG4, human IgG4 (S228P), human IgG2, human IgG1, or human IgG1 effector-invalidated region. For example, the human IgG4 (S228P) Fc region, compared to the wild-type human IgG4 Fc region, includes the following substitution: S228P. For example, the human IgG1 effector-invalidated Fc region, compared to the wild-type human IgG1 Fc region, includes the following substitutions: 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:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0092] This document provides an immunoconjugate comprising a disclosed protein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell-inhibiting enzyme, a cell-lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0093] Examples of suitable therapeutic agents include, but are not limited to, immunomodulators, cytotoxins, radioisotopes, chemotherapeutic agents, anti-angiogenic agents, antiproliferative agents, pro-apoptotic agents, and cell-inhibiting and cell-lysing enzymes (e.g., RNases). Other therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulators, anti-angiogenic agents, antiproliferative agents, or pro-apoptotic agents. These drug descriptors are not mutually exclusive, and therefore one or more of the terms above may be used to describe a therapeutic agent.

[0094] Examples of suitable therapeutic agents for 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 antifolate agents, vinca alkaloids, and anthracyclines. Phytotoxins, other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, and photosensitizers can also be used for immunoconjugates. Furthermore, secondary carriers (such as liposomes or polymers) can be used as cytotoxic agents to prepare conjugates. Suitable cytotoxins include agents that inhibit or prevent cell function and / or cause cell destruction. Representative cytotoxins include antibiotics, microtubule polymerization inhibitors, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins (such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins).

[0095] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carrubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, and gemcitabine. Itabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin The following are listed: tegafur, tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, and methotrexate. Fluorouracil, etoposide, taxol, taxol analogs, platinum-based drugs (such as cisplatin and carboplatin), mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, autramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins / olestradiol, hemiasterlinEsperamicins and maytansinoids.

[0096] Suitable immunomodulators include antihormonal drugs that block the effects of hormones on tumors and immunosuppressants that inhibit cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0097] Table 5: Strand sequence combinations for exemplary proteins

[0098] Pharmaceutical Composition The activatable proteins (also referred to herein as “active compounds”) described herein can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise a protein (or an immunoconjugate containing said protein) and a pharmaceutically acceptable carrier, diluent, or excipient. Such materials should be non-toxic and should not interfere with the efficacy of the protein. The exact properties of the carrier or other materials will depend on the route of administration, which may be by injection, pellet, infusion, or any other suitable route, as discussed below.

[0099] As used herein, the term "pharmaceuticalally acceptable" refers to molecular entities and compositions that, when administered using methods well known in the art, do not typically cause allergic reactions or other serious adverse reactions. Molecular entities and compositions approved for use in animals (especially humans) by U.S. federal or state regulatory agencies or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias are considered "pharmaceuticalally acceptable." As used herein, the term "pharmaceuticalally acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences (the standard reference text in the art), which is incorporated herein by reference. Some examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous media, such as non-volatile oils, may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or reagents are incompatible with the active compound, their use in the composition is contemplated. Additional active compounds may also be incorporated into the composition. Pharmaceutically acceptable carriers, diluents, or excipients may be compounds or combinations of compounds that do not cause secondary reactions and allow, for example, to facilitate protein administration, prolong its shelf life, and / or enhance its efficacy in vivo or increase its solubility in solution.

[0100] The pharmaceutical compositions disclosed herein can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, such as water for injection, saline solutions, non-volatile oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfate; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or glucose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0101] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (in the water-soluble case) or dispersants and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersants. For intravenous administration, suitable carriers include physiological saline, antibacterial aqueous solutions, and Cremophor EL. ® (BASF, Parsipani, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to a degree of ease of injection. It must be stable under manufacturing and storage conditions; and must be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, such as containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating (such as lecithin), maintaining the desired particle size in the case of a dispersant, and by using a surfactant. Microbial action can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include an isotonic agent in the composition, such as sugars, polyols (such as mannitol, sorbitol), or sodium chloride. Extended absorption of the injectable composition can be achieved by including a delayed absorption agent (e.g., aluminum monostearate and gelatin) in the composition.

[0102] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound, along with one or more of the components listed above (as needed), into a suitable solvent, followed by filtration and sterilization. Typically, dispersants are prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium and other desired components from those listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying to obtain a powder of the active ingredient plus any other desired components from a previously sterile filtered solution.

[0103] Oral compositions typically include inert diluents or edible carriers. They can be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with excipients and administered in tablet, lozenge, or capsule form. Oral compositions can also be prepared using fluid carriers for use as mouthwashes, wherein the compound in the fluid carrier is applied orally and rinsed and spat out or swallowed. Pharmaceutically compatible binders and / or excipients can be included as part of the composition. Tablets, pills, capsules, lozenges, etc., can contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primojel ® Or corn starch; lubricants, such as magnesium stearate; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0104] For inhalation administration, the compound can be delivered as an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant (e.g., a gas such as carbon dioxide).

[0105] Systemic application can also be performed via mucosal or transdermal routes. For mucosal or transdermal application, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are well known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives used for mucosal application. Mucosal application can be accomplished by using nasal sprays or suppositories. For transdermal application, the active compound is formulated as an ointment, cream, gel, or lotion well known in the art.

[0106] The medication can also be formulated as a suppository (e.g., using a conventional suppository base, such as cocoa butter and other glycerides) or as a retention enema for rectal delivery.

[0107] In some embodiments, the active compound is prepared together with a carrier (such as a controlled-release formulation, including implants and microencapsulated delivery systems) that protects the compound from rapid elimination from the body. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be readily apparent to those skilled in the art. The materials are also commercially available. Liposome suspensions can also be used as pharmaceutically acceptable carriers.

[0108] Formulating oral or parenteral compositions in dosage units is particularly advantageous because it facilitates administration and ensures uniform dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for use on the subject to be treated; each unit contains a predetermined amount of active compound calculated to associate with the desired drug carrier to produce the desired therapeutic effect. The specifications of the dosage unit form of the present invention are determined by and directly depend on the unique properties of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the prior art of synthesizing such active compounds for treating individuals.

[0109] In some implementations, proteins 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 before administration to an individual.

[0110] The pharmaceutical compositions described herein may be included in containers, packages or dispensers together with the instructions for use.

[0111] Nucleic acid molecules, vectors, host cells, and methods for producing proteins This document provides a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding the amino acid sequence of the protein disclosed herein (or the amino acid sequence of (i) the VH domain, (ii) the VL domain, or (iii) both the VH and VL domains of the protein). This document further provides a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding (i) the heavy chain, (ii) the light chain, or (iii) both the heavy and light chains of the protein disclosed herein. In some embodiments, the nucleic acid molecule encoding the VH domain, VL domain, heavy chain, or light chain includes a signal sequence (or encodes a leader peptide). In some embodiments, the nucleic acid molecule encoding the VH domain, VL domain, heavy chain, or light chain does not include a signal sequence (or does not encode a leader peptide).

[0112] This document also provides expression vectors comprising the nucleic acid molecules described herein. In some vectors, the nucleic acid molecule is operatively linked to one or more regulatory sequences suitable for expression of the nucleic acid segment in a host cell. In some cases, the expression vector contains a replication-mediating sequence and includes one or more optional markers. As used herein, “vector” means a construct capable of delivering and preferably expressing 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, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0113] This document provides a recombinant host cell comprising the expression vector or nucleic acid molecule disclosed herein. "Host cell" includes an individual cell, cell line, or cell culture that may be or has been a recipient of a vector for incorporating a polynucleotide intercalation. Host cells include progeny of a single host cell. Progeny may not necessarily be identical to the original parent cell (morphologically or in terms of genomic DNA complementarity) due to natural, accidental, or intentional mutations. Expression vectors can be transfected into host cells using standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc. In some embodiments, the recombinant host cell comprises a single vector or a single nucleic acid molecule encoding both the heavy and light chains of the protein disclosed herein. In some embodiments, the recombinant host cell comprises (i) a first vector or a first nucleic acid molecule encoding the heavy chain of the protein disclosed herein and (ii) a second vector or a second nucleic acid molecule encoding the light chain of the protein disclosed herein.

[0114] The protein molecules or portions thereof of the present invention may be produced using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, computational techniques, or combinations thereof, or other techniques readily known in the art.

[0115] This document further provides a method for producing the protein disclosed herein, the method comprising: culturing a recombinant host cell containing the expression vector described herein under conditions where a nucleic acid fragment can be expressed, thereby producing the protein. The protein can then be isolated from the host cell or culture. This document provides a method for producing a protein, the method comprising: culturing a recombinant host cell containing the expression vector disclosed herein under conditions where a nucleic acid molecule can be expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0116] The proteins disclosed herein can be produced by any of a variety of methods known to those skilled in the art. In some embodiments, the proteins disclosed herein can be produced by recombinant methods. For example, a nucleic acid sequence or a portion thereof encoding one or more of the heavy or light chains provided herein can be introduced into a bacterial cell (e.g., *Escherichia coli*). E. coli Bacillus subtilis ( B. subtilis or eukaryotic cells (e.g., yeast, such as Saccharomyces cerevisiae). 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 introduced into an in vitro translation system, and the translated polypeptide can be isolated. In some embodiments, light chain proteins and heavy chain proteins are generated in cells with a signal sequence that is removed during the production of the mature protein disclosed herein.

[0117] Those skilled in the art can use standard methods, such as Western blotting, ELISA, etc., to determine whether a protein containing a given polypeptide sequence binds to the PD-L1 protein and / or the CD28 protein.

[0118] Medical uses of proteins that can be activated This document provides methods and uses of the disclosed methods for activating proteins, immune conjugates, and pharmaceutical compositions for providing therapeutic benefits to subjects with cancer.

[0119] The activatable proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can be used in methods of treating humans or animals, including preventative or prophylactic treatments (e.g., treating a subject before the onset of a condition to reduce the risk of the condition occurring in the subject; delaying its onset; or reducing its severity after onset). Treatment methods may include administering the protein, immunoconjugate, or pharmaceutical composition to the subject in need.

[0120] This document provides a method for enhancing an anticancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the disclosed protein, immunoconjugate, or pharmaceutical composition. In some embodiments, the anticancer immune response is a T-cell response. In some embodiments, the anticancer immune response is a complement response.

[0121] This article provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0122] This article provides a method for alleviating cancer symptoms in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0123] This article provides a method 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.

[0124] This article provides a method for inhibiting tumor growth in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0125] In some implementation schemes, cancer is gastrointestinal stromal carcinoma (GIST), pancreatic cancer, skin cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, prostate cancer, stomach cancer, ovarian cancer, bladder cancer, brain cancer, central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine cancer, endometrial cancer, cancer of the mouth or throat, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, bile duct cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy.

[0126] In some implementations, the cancer is a solid tumor. In some implementations, the cancer is a blood cancer.

[0127] In some implementations, blood tissue cancer is lymphoma. In some implementations, cancer is mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, acute myeloid leukemia (AML), B-cell lymphocytic leukemia, blastic plasmacytoid dendritic neoplasm (BPDCN), or hairy cell leukemia.

[0128] As used herein, the terms “effective amount” or “therapeutic effective amount” refer to an amount of the agent (e.g., protein, immune conjugate, or pharmaceutical composition) disclosed herein that is sufficient to reduce or alleviate the severity and / or duration of cancer, or one or more symptoms thereof, prevent the development of the disease, induce the remission of the disease, prevent the recurrence, development, onset, or progression of one or more symptoms associated with the disease, or enhance or improve the preventive or therapeutic effect of another related therapy (e.g., a preventive or therapeutic agent) on cancer.

[0129] The actual dosage, rate, and timing of administration will depend on the nature and severity of the disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the composition, the method of administration, the timing of administration, and other factors known to the practicing physician. Treatment prescriptions, such as dosage decisions, fall under the responsibility of general practitioners and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated. Appropriate dosages of antibody-based protein molecules are well-known in the art (Ledermann JA et al., 1991). Int. J. Cancer47: 659-664; Bagshawe KD et al., 1991, Antibody , Immunocouples and Radiopharmaceuticals 4: 915-922). Specific dosages can be found in this article or in [the following text is missing from the original] Physician's Desk Reference (2003) states that the appropriate dosage should be determined based on the type of drug that may be used. The therapeutically effective or appropriate dose of an antibody-based protein molecule can be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective doses from mice and other test animals to humans are known. The precise dosage will depend on a number of factors, including whether the antibody-based protein is used for prevention or treatment, the size and location of the area to be treated, the precise nature of the antibody-based protein, and the nature of any detectable markers or other molecules attached to the antibody-based protein.

[0130] For systemic application, typical protein doses range from 100 μg to 1 g, and for intradermal injection, from 1 μg to 1 mg. A higher initial loading dose may be administered, followed by one or more lower doses. In some embodiments, the protein is an IgG1 or IgG4 isotype. The single treatment dose for adult subjects may be proportionally adjusted for use in children and infants. Treatment may be repeated at daily, twice-weekly, weekly, or monthly intervals, as determined by the physician. The timing of treatment for a subject may depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated.

[0131] Treatment can be periodic, and the time intervals between applications can be approximately two weeks or longer, such as approximately three weeks or longer, approximately four weeks or longer, approximately once a month or longer, approximately five weeks or longer, or approximately six weeks or longer. For example, treatment can be administered every two to four weeks or every four to eight weeks. Treatment can be given before and / or after surgery, and / or can be applied directly to the anatomical site of the surgical treatment or invasive procedure. Suitable formulations and routes of administration are as described above.

[0132] In some embodiments, the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can be administered via subcutaneous injection. Subcutaneous injection can be administered using an autoinjector, for example, for long-term prevention / treatment.

[0133] In some embodiments, the therapeutic effect of the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can last for several half-lives, depending on the dose. For example, a single dose of the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can last in the subject for one month or longer, two months or longer, three months or longer, four months or longer, five months or longer, or six months or longer.

[0134] In some embodiments, a subject may be treated with the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein, along with adjunctive therapeutic agents or therapies for treating cancer or cancer symptoms or complications. The proteins, immunoconjugates, or pharmaceutical compositions disclosed herein, along with adjunctive therapeutic agents or therapies, may be administered simultaneously or sequentially.

[0135] In some implementations, 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 implementations, the subject is an adult. In some implementations, the subject is a child.

[0136] This article further provides the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein for the treatment of diseases or conditions.

[0137] This document provides the proteins, immunoconjugates, or pharmaceutical compositions disclosed herein for use as medicines. definition

[0138] Unless otherwise specified, the terms used herein have their definitions as commonly used in the art. Some terms are defined below, and others can be found in the remaining detailed description.

[0139] The term “a” or “one (species)” refers to one or more (species) of the entity, that is, it can refer to a plural indicator. Therefore, the terms “a,” “one (species),” “one (species) or more (species),” and “at least one (species)” are used interchangeably in this document. Furthermore, using the indefinite article “a” or “one (species)” to refer to “one (species) element” does not preclude the possibility of more than one (species) element, unless the context explicitly requires the existence of one (species) and only one (species) element.

[0140] Unless otherwise stated or the context clearly indicates otherwise, the term "about" means within 10% of the reported value (unless such a figure would exceed 100% of the possible value or be less than 0%). When used with a range or series of values, the term "about" applies to the endpoints of the range or each of the values ​​enumerated in the series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0141] As used herein, the term “sequence identity” refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences remain unchanged throughout the entire residue (e.g., nucleotide or amino acid) alignment window. The “identity score” of the aligned segments of the test and reference sequences is the number of common residues in 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). “Percentage identity” is the identity score multiplied by 100. Percentage identity can be calculated using the alignment program Clustal Omega, which can be obtained at ebi.ac.uk / Tools / msa / clustalo with default parameters. See Sievers et al., “Fast, scalable generation of high-quality protein multiplesequence alignments using Clustal Omega” (October 11, 2011). Molecular Systems Biology 7:539. For the purpose of calculating sequence identity, extensions such as labels are not included.

[0142] As used herein, the term "HCDR" refers to the heavy chain complementarity-determining region. As used herein, the term "LCDR" refers to the light chain complementarity-determining region.

[0143] As used herein, the terms “amino terminus,” “N-terminus,” “carboxyl terminus,” and “C-terminus” refer to positions within the polypeptide chain. Where the context permits, these terms are used to indicate proximity or relative position for a specific sequence or portion of a reference polypeptide. For example, a sequence located at the carboxyl terminus of a reference sequence within a polypeptide may be located proximal to the carboxyl terminus of the reference sequence, but not necessarily at the carboxyl terminus of the intact polypeptide.

[0144] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid without significantly and harmfully altering its functional activity. A preferred example of "conservative substitution" is the replacement of one amino acid with another amino acid having a value ≥0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992). PNAS 89: 10915-10919): .

[0145] The term "immunoconjugate" refers to a protein of this disclosure conjugated with a cytotoxic agent, cell inhibitor, and / or therapeutic agent.

[0146] The term "isolated molecule" (in the case of molecules such as proteins, nucleic acids, polynucleotides, or antibodies) is defined as a molecule that, based on its origin or derived source, (1) does not associate with its naturally associated components in their native state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) is not present in nature. Therefore, a chemically synthesized molecule or a molecule expressed in a cellular system different from the cells of its natural origin will be "isolated" from its naturally associated components. The molecule can also be made substantially free of its naturally associated components by isolation using purification techniques well known in the art. Molecular purity or homogeneity can be determined in a variety of ways well known in the art. For example, the purity of a peptide sample can be determined by using polyacrylamide gel electrophoresis and gel staining to visualize the peptide using techniques well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other purification methods well known in the art.

[0147] As used herein with respect to the biological activity of the proteins disclosed herein, the terms “inhibition,” “blocking,” or “neutralization” refer to the ability of a protein to significantly antagonize, prohibit, prevent, limit, slow down, disrupt, eliminate, stop, reduce, or reverse, for example, the progression, intensity, or severity of something that is inhibited (including, but not limited to, the binding of PD-L1 to PD-1 or the binding of CD28 to CD80 and / or CD86).

[0148] As used herein, the terms “treat,” “treating,” or “therapeutic” (and their grammatical variations) refer to a reduction, at least partial improvement, or stabilization of the severity of the subject condition, and / or a certain degree of relief, reduction, decrease, or stabilization of at least one clinical symptom, and / or a delay in the progression of the disease or condition.

[0149] PD-L1 is also known as programmed cell death ligand 1, CD274, B7-H, B7H1, PDCD1L1, PDCD1LG1, PDL1, and hPD-L1.

[0150] CD28 is also known as differentiation cluster 28 and TP44. CD28 is a type I transmembrane protein.

[0151] As used herein, the terms “prevent,” “preventing,” and “prevention” (and their grammatical variations) refer to preventing and / or delaying the onset of a disease, condition, and / or clinical symptom in a subject and / or reducing the severity of the onset of a disease, condition, and / or clinical symptom compared to the absence of the compositions and / or methods described herein. Prevention can be complete, for example, the complete absence of a disease, condition, and / or clinical symptom. Prevention can also be partial, such that the occurrence and / or severity of a disease, condition, and / or clinical symptom in the subject is less than that would occur in the absence of the compositions and / or methods described herein.

[0152] As used herein, "therapeutic effective amount" is the amount of protein or pharmaceutical composition provided herein that is effective in treating or preventing a disease or condition in a subject or in alleviating its signs or symptoms. "Therapeutic effective amount" may vary, for example, depending on the disease and / or the symptoms of the disease, the severity of the symptoms of the disease and / or condition, the age, weight and / or health status of the patient to be treated, and the judgment of the prescribing physician.

[0153] As used herein, unless otherwise stated, the terms “hinge connector,” “joint,” “hinge,” “first connector,” “second connector,” “lower hinge connector” (“LHL”), “Fab indirect connector,” and their derivatives, in plural or singular form, refer to sequences that can connect two polypeptides (e.g., polypeptides from different Fab regions), such as sequences derived from immunoglobulin hinge regions, and are separate from any hinge sequences in the immunoglobulin hinge region that may be part of the proteins of the present invention.

[0154] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated in their entirety for all purposes. However, any mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be construed as, an admission or in any way an implication that they constitute valid prior art or are part of common general knowledge in any country of the world.

[0155] The chapter titles used in this article are for organizational purposes only and should not be considered as limiting the topics described.

[0156] This disclosure will be further illustrated by the following embodiments, but these embodiments are intended to illustrate the disclosure only and are not intended to be limiting. Example

[0157] This disclosure will be illustrated in more detail through the following embodiments. However, it should be understood that this disclosure is not limited in any way to these embodiments.

[0158] The embodiments of this disclosure describe the generation and optimization of the conditionally active therapeutic proteins disclosed herein.

[0159] Example 1: Materials and Methods Protein cloning, transient expression, purification and characterization The DNA sequence encoding the polypeptide was cloned via restriction-ligation cloning into an expression cassette encoding the individual human IgG1 heavy and light chain constant regions in a separate plasmid vector to create an activatable construct for expression. The protein was expressed in CHO cells and purified from the culture supernatant by a combination of protein A affinity chromatography (ProA), ion exchange chromatography (IEX), and / or size exclusion chromatography (SEC). The purified protein was characterized by SEC, SDS-PAGE, and mass spectrometry.

[0160] Metalloproteinase digestion The protein constructs were incubated with full-length human matrix metalloproteinase (MMP) enzymes (LPD28-1 and LPD28-2) or those catalyzing only the MMP12 domain (LPD28-3, -4, -5, -6, 7, -8, -9, -10, -11, -12, -13) at 37°C in time increments of 0 to 24 hours. The MMP12 enzymes were added to Tris-buffered saline (pH 7.4) containing 5 mM CaCl2 at a ratio of 1% total MMPs to protein constructs (wt / wt). The reaction was terminated by adding 20 mM EDTA, and the binding or functional activity of the samples was then tested as described.

[0161] Surface plasmon resonance of Fab protein To evaluate the binding of assay Fab to human or mouse CD28 (Acro Biosystems, Newark, USA), multi-cycle kinetic analysis was performed at 25°C on a Biacore® 8K (serial number 2724204). HBS-P+ (Cytiva, Marlborough, USA) supplemented with 0.1% BSA (Sigma, Dorset, UK) was used as the run buffer and ligand and analyte dilution. CD28 protein was diluted to 1.0 μg / mL in the run buffer and loaded at the start of each cycle onto the F-array of an S-series CM5 chip (Cytiva, Marlborough, USA) previously conjugated with an anti-His capture antibody (Cytiva, Marlborough, USA) using standard amine chemistry. c2. Ligands were captured at a flow rate of 10 μl / min to achieve an immobilization level (RL) of approximately 60 RU. The surface was then stabilized. Antigen was injected as analyte at a flow rate of 30 μl / min to obtain multi-cycle kinetic data to minimize any potential mass transfer effects. Nine 2-fold dilutions of purified Fab, titrated from 0.39 nM to 100 nM, were prepared in Fab protein run buffer. For each concentration, the binding phase was monitored for 240 seconds, and the dissociation phase was measured for 900 seconds. The sensor chip surface was regenerated between cycles using 10 mM glycine at pH 2.1. Multiple repetitions of blank and antigen were programmed into the kinetic run to examine the stability of both the surface and the analyte during kinetic cycling. Reference F c The signal of 1 (uncaptured ligand) from F c The signal from the 2-channel test is subtracted to correct for volume effects and differences in nonspecific binding to the reference surface. The signal from each blank run (capturing ligand but without antigen) is subtracted to correct for differences in surface stability. Due to the high affinity interaction between the antibody and antigen, a 1:1 binding assay is used to analyze binding. A procedure called dual-reference is used to analyze the data. This refers to first subtracting the reference channel response (F... c 1), and then the process of subtracting the zero-concentration sensor map to compensate for volume effects, baseline drift, and minor differences between the reference and active channels.

[0162] T cell activation bioassay The functional activity of the protein construct was assessed in a co-culture assay using MDA-MB-231 (PD-L1 High) and the Jurkat reporter cell line (Promega – IL2 promoter). MDA-MB-231 (PD-L1 High) cells (500,000 cells / well for 96 wells or 50,000 cells / well for 384 wells) were seeded into 96-well or 384-well white-bottomed tissue culture 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 either the control antibody or the protein construct (+ / - pre-digested with MMP3 / 7 / 12) prepared in assay medium (RPMI supplemented with 10% FBS) was added to the cells. Jurkat effector cells (IL2) were thawed and diluted according to the manufacturer's protocol and then added to the assay wells. After incubating at 37°C for 6 hours in a CO2 incubator, the plate was reequilibrated to room temperature, and luciferase activity was determined by adding Bio-Glo reagent for 5–10 minutes and measuring the luminescence signal (RLU). The fold induction was determined by calculating the RLU / RLU ratio of the sample without antibody after subtracting the background luminescence signal.

[0163] ELISA binding of IgG and LB proteins For ELISA binding assays, coat 384-well or 96-well plates with 1 μg / ml and incubate overnight at 4°C in the dark. Wash the plates twice with PBS / 0.05% Tween 20, then block with blocking buffer (3% lactic acid in PBS) for 1 hour at room temperature. Next, load the test reagent or control and incubate for 1 hour at room temperature. Then wash the plates three times with PBS / 0.05% Tween 20. Finally, add 1 / 5,000 HRP-conjugated secondary antibody diluted in blocking buffer and incubate for 1 hour at room temperature. Wash the plates three more times with PBS / 0.05% Tween, then add TMB substrate and incubate for 6 minutes at room temperature. Finally, terminate the reaction with stop solution. Read the absorbance at 450 nm and 570 nm. Subtract the absorbance at 570 nm from the absorbance at 450 nm for each plate.

[0164] Cellular binding of LB protein Jurkat cells were harvested, counted, and washed once with PBS. They were then stained with Zombie UV-active dye diluted 1:1000 in PBS at room temperature (RT) for 30 min. Induction was determined by calculating the RLU / RLU ratio of the sample without antibody after subtracting the background luminescence signal. Cells were washed with staining buffer (0.1% BSA in PBS), resuspended in the staining buffer, and separated into test aliquots. Cells were incubated with LB protein on ice for 30 min at the specified time. Cells were washed twice with the staining buffer and then incubated on ice with secondary antibody or staining buffer alone for 60 min. After incubation with secondary antibody, cells were washed twice with the staining buffer and then fixed with 4% PFA at room temperature for 10 min. Fixed cells were resuspended in the staining buffer and stored at 4°C for analysis using a BD LSR Fortessa flow cytometer.

[0165] MLR measurement PBMCs were removed from cryogenic storage and thawed in serum-free medium. Monocytes were isolated by positive magnetic separation and seeded at 2 x 10⁶ cells / mL in CellGenix DC medium supplemented with gentamicin (50 μg / mL), human granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 μg / mL), and human interleukin-4 (IL-4) (100 μg / mL). Cells were allowed to differentiate into iDCs at 37°C and 5% CO₂ for five days. On day 5, MDA-MB-231 cells were harvested and irradiated with 25 Gy. After irradiation, cells were washed three times with total RPMI. Cells were then counted and seeded at 10,000 cells / well in 96-well plates and incubated at 37°C and 5% CO₂ for at least 1 h. Also on day 5, fresh PBMCs were removed from cryogenic storage and thawed in serum-free medium. Allogeneic CD3+ T cells were isolated using negative magnetic separation. iDCs were harvested and co-cultured with CD3+ T cells at a 1:10 ratio in total RPMI in 96-well plates containing MDA-MB-231 cells, with or without the test compound and its corresponding control. The co-cultures were incubated at 37°C and 5% CO2 for 2 to 5 days. After incubation with the test and / or control compounds for two days (IL-2 measurement) and five days (IFN-γ measurement), the plates were centrifuged at 800 g for 5 min at 4°C, and the supernatant was transferred to V-plates. These plates were then centrifuged at 800 g for 5 min at 4°C, and the supernatant was transferred to new V-plates. The plates were sealed and stored at -80°C until further use. IL-2 and IFN-γ concentrations in the supernatant were measured by ELISA using the LEGEND MAX™ Human IL-2 Kit and LEGEND MAX™ Human IFN-γ Kit, respectively, through pre-coated plates, following the manufacturer's instructions.

[0166] Primary T-cell killing MDA-MB-231 Tumor cells were counted and seeded into appropriate 96-well plates and allowed to adhere for approximately 24 h. PBMCs were isolated from whole blood by centrifugation, followed immediately by T cell isolation. For this purpose, PBMCs were incubated with a mixture of Pan T cell microbeads and then applied to a MACS separation column. Flow-through cells representing enriched T cells were collected and diluted to the desired levels. For the Incucyte® live cell analysis platform, LPD protein or buffer stained with an appropriate concentration of Annexin V was added to the adhered tumor target cells, followed immediately by the addition of T cells, and the plate was placed in an Incucyte® S3 live cell analysis system incubator. Scan intervals were set every 4 h for 150 h. Annexin V data were normalized to buffer / T cell / tumor cell signal prior to plotting.

[0167] Cytokine release assay - solid phase assay The provided test samples and two controls were used for plate binding assays at 1 μg / well. PHA (Sigma Aldrich, Poole, UK) and anti-CD3 (OKT3) (Biolegend, San Diego, USA) were used as positive controls (final assay concentrations were 10 μg / mL and 1 μg / well, respectively).

[0168] A negative control culture was established, in which PBMC cultures were treated with PBS. Clinical antibodies TGN1412 (Evitria, Zurich, Switzerland) and Erbitux® (Eli Lilly, Indianapolis, USA) were used with a final assay coating density of 1 μg / well. 60 μL of each antibody control or sample was added to two copies of each 96-well polypropylene plate (Corning, Amsterdam, The Netherlands) and incubated at room temperature for 2 hours. After incubation, the plates were washed with 2 x 200 μL PBS, followed by 200 μL of culture medium to remove any unbound antibodies. 100 μL of culture medium was added to each well, or PHA-containing medium was added to the corresponding control well, along with 100 μL of freshly isolated PBMCs at 2 x 10⁶ cells / mL. The plates were then incubated at 37°C and 5% CO₂ (method based on the method used by Eastwood et al., 2013). After 72 hours of incubation, the supernatant was removed from the culture and stored at -80 °C until use. Cytokine levels in the supernatant were analyzed using a magnetic Milliplex kit (Merck Millipore, Hertfordshire, UK) according to the manufacturer's instructions. The supernatant, serially diluted five-fold standards, and mass controls in the culture medium were incubated with premixed capture beads for 16–18 hours before being incubated with the assay reagent. Data were acquired using FLEXMap 3D® (Bio-Rad, Hertfordshire, UK). Cytokine secretion levels (in pg / ml) were determined using a standard curve with xPONENT® software (Bio-Rad). The mean values ​​of the sample and control replicates were used in the analysis, with LQC and HQC (determined as the lowest and highest quantifiable calibrators) used as limits of detection; any values ​​below or above these limits were replaced by LQC and HQC values, respectively.

[0169] PBMC cytokine release assay Frozen PBMCs were thawed, counted, and resuspended in culture medium to the appropriate concentration. Next, cells were seeded at 2 x 10⁵ cells per well into 96-well round-bottom plates containing a 5-fold concentrated test sample and controls. Cells were cultured at 37°C and 5% CO₂ for 24 h. After 24 h, the plates were centrifuged at 300 g for 10 min at room temperature (RT), and the supernatant was collected and transferred to new 96-well, non-sterile, V-bottom plates. Each plate was centrifuged twice at 800 g for 3 min at room temperature, and the supernatant was again collected and transferred to different 96-well, non-sterile, V-bottom 96-well plates and stored at -80°C until further analysis. Cytokine release assay: Solubility incubation of the test reagent.

[0170] PBMC recovery cytokine release assay.

[0171] Frozen PBMCs were thawed, counted, and resuspended in culture medium to the appropriate concentration. Next, cells were seeded at 1.5 x 10⁷ cells per well in 24-well plates and incubated at 37°C and 5% CO₂ for 48 h. After pre-incubation, PBMCs were harvested, counted, and seeded at 2 x 10⁵ cells per well in 96-well round-bottom plates containing a 5-fold concentrated test sample and controls. Cells were maintained at 37°C and 5% CO₂ for 24 h. After 24 h, the plates were centrifuged at 300 g for 10 min at room temperature (RT), and the supernatant was harvested and transferred to new 96-well, non-sterile, V-bottom plates. Each plate was centrifuged twice at 800 g for 3 min at RT, and the supernatant was harvested again and transferred to different 96-well, non-sterile, V-bottom 96-well plates and stored at -80°C until further analysis. Cytokine release assay: Solubility incubation of the test reagent.

[0172] Example 2: Results and Discussion Protein construct design, cloning, expression, and characterization To generate proteins for functional testing, DNA cassettes for each construct type were designed using combinations of variable domain, constant domain, and adapter sequences from Table 1. These CD28 humanized variants were then bound to PD-L1 variable domains, constant domains, and adapters to form full-length heavy and light chain sequences (Table 4). Using these full-length chains, two initial designs were synthesized and cloned into expression vectors encoding human IgG1 heavy and light chain sequences as well as a free hinge-Fc fragment (asymmetric one-arm construct) (Table 5 and Figure 1). Additional designs were synthesized and cloned into expression vectors encoding human IgG1 heavy and light chain sequences to create symmetric one-arm or two-arm constructs similar to the more classic IgG phenotype (Table 5, Figure 1). Figure 1A And B). Two CD28 antibody sequences were expressed in Fab form, referred to as Fab_001 (CD28 TGN-derived sequence) and Fab_002 (Table 2), and in IgG form, referred to as IgG_001 and IgG_002 (Table 3). Binding of Fab_001 and Fab_002 to CD28 was tested in surface plasmon resonance (SPR). Figure 3 Fab_001 was shown to bind to recombinant human / cynomolgus monkey CD28 with a high affinity of 0.59 nM (KD). Fab_002 showed a similar high affinity (KD 0.53 nM) for recombinant human / cynomolgus monkey CD28 protein. Neither protein bound to mouse CD28 protein as expected. The binding of IgG_001 and IgG_002 to coated human / cynomolgus monkey CD28 protein was subsequently tested in an ELISA. Figure 4 As shown in the Biacore experiment, both IgG_001 and IgG_002 exhibited high binding affinity similar to human / cynomolgus monkey CD28, but showed no binding with mouse CD28. The IgG1 isotype control did not show binding. TGN1412 (IgG4 isotype) showed overlapping binding characteristics with IgG_001, confirming that the removal of unpaired cysteine ​​residues and the IgG1 isotype did not affect the binding affinity of IgG_001.

[0173] Next, the LPD proteins described in Table 5 were prepared by transient transfection of CHO cells and then purified by ProA, IEX, and / or SEC. Following the ProA step, protein yield and homogeneity were assessed by SEC (Table 6). The fully purified proteins exhibited high purity (>95%) and homogeneity upon SEC analysis, indicating that the best-performing constructs can be expressed and purified into intact, stable products using a single method. The symmetric single-arm constructs showed significantly lower purity (LPD28-6 to LPD28-9, Table 6) after the initial ProA purification step.

[0174] Table 6: Productivity Analysis of Selected Proteins

[0175] HMW = High molecular weight products LMW = Low molecular weight products In the presence or absence of human MMP12 enzyme, homogeneous and completely purified molecules were incubated at 37°C for a specified time, and the digestion profiles were evaluated using SDS-PAGE under both reducing and non-reducing conditions. Figures 5A to 5H ). Figure 5A Figures B and B show the digestion profiles of exemplary asymmetric single-arm LPD proteins (LPD28-1, LPD28-2, and LPD28-3). LPD28-1 and LPD28-2 were treated with a full-length MMP12 enzyme, while LPD28-3 and subsequent molecules were digested with a catalytic domain MMP12 enzyme. In both reducing and non-reducing SDS-PAGE, the undigested (intact) proteins showed the expected banding pattern, with a prominent band at 150 kDa visible in the non-reducing gel, and three chains at 75 kDa (LB heavy chain), 50 kDa (LB light chain), and approximately 30 kDa (Fc residues) visible in the reducing gel. Figure 5A(and B). After incubation with MMP12 at 37°C, the band distribution was significantly different from that of the undigested protein, indicating that both the LB heavy chain and the LB light chain were cleaved. In the reducing SDS-PAGE, multiple 25-30 kDa bands appeared (corresponding to the VC domain cleaved from the LB light or heavy chain, and the single-chain Fc fragments released by protein hydrolysis) and approximately 55 kDa bands (corresponding to the VH-CH domain with Fc), and the corresponding disappearance of the intact LB light and heavy chain products at 75 and 50 kDa, indicating that the chains were cleaved at their LHL linkers, resulting in the formation of products such as... Figure 5A and Figure 5B The corresponding products are shown.

[0176] Further MMP12 enzyme digestion and SDS-PAGE analysis of other LPD protein patterns ( Figure 5C -H) showcases different patterns, reflecting their respective designs: symmetric single-arm construct digestion such as Figure 5C As shown in Figures D and E, the symmetrical two-armed LPD protein is as follows: Figure 5F As shown in Figures G and H, the undigested symmetrical single-arm LPD protein exhibited the expected banding pattern in both reduced and non-reduced SDS-PAGE, with a prominent band at 150 kDa visible in non-reduced SDS-PAGE. Consistent with expectations, in the reduced SDS-PAGE gel, only one chain was visible at 75 kDa compared to the asymmetric LPD molecules (LB heavy chain and LB light chain). Figure 5C (D, E). After incubation with MMP12 at 37°C, the band distribution was significantly different from that of the undigested protein, indicating that the 75 kDa band was cleaved. In reducing SDS-PAGE, the appearance of 25-30 kDa bands corresponded to the VC domains cleaved from the LB light or heavy chains. Additionally, a band of approximately 55 kDa appeared, corresponding to the VH-CH and VL-CH domains with Fc. The undigested (intact) LPD light and heavy chain products at 75 kDa disappeared with increasing MMP12 treatment time, indicating that either chain of both LHLs was cleaved, resulting in the VC domains cleaved from the LB light or heavy chains. Figure 5C The corresponding products shown in D and E. Figure 5C The selected symmetrical single-arm LPD proteins shown were designed to contain different hinge heads: LHL and LHL-F combination (LPD28-6) or LHLX and LHLX-F combination (LPD28-7). Figure 5C In the symmetrical single-arm form, LPD proteins containing either the LHL and LHL-F combination (LPD28-6) or LHLX-LHLX-F (LPD28-7) showed no significant difference in sensitivity to MMP12 cleavage. Figure 5C ).Will Figure 5DThe selected symmetrical single-arm LPD protein shown is designed as a combination of LPD hinge heads LHL and LHL-FR, wherein LHL-FR appears in either the VH chain (LPD28-8) or the VL chain (LPD28-9). Figure 5D The LHL-FR design theoretically demonstrates higher sensitivity to MMP cleavage. In the symmetrical single-arm form, LPD proteins containing LHL-FR in the VL or VH chain showed no significant difference in sensitivity to MMP12 cleavage. Figure 5D ).

[0177] Undigested biarm LPD protein showed the expected banding pattern in both reduced and non-reduced SDS-PAGE, with a prominent band at 250 kDa visible in the non-reduced gel and two chains at 75 kDa (LB heavy chain) and 50 kDa (LB light chain) visible in the reduced gel. Figure 5F (G, H). After incubation with MMP12 at 37°C, the band distribution was significantly different compared to intact / undigested proteins, indicating that both LPD heavy and light chains were cleaved. In reductive SDS-PAGE, the appearance of multiple 25-30 kDa bands corresponded to the VC domain cleaved from either the LPD light or heavy chain. In reductive SDS-PAGE, the appearance of approximately 55 kDa bands corresponded to the VH-CH domain with Fc. Undigested LPD light and heavy chain products at 75 kDa and 50 kDa disappeared with increasing MMP12 treatment time, indicating that either intact chain of both LHLs was cleaved, resulting in products such as... Figure 5F , 5G The corresponding products shown in 5H. Figure 5F and Figure 5G A two-armed LPD protein was described, designed with an LPD linker LHL in its heavy chain and LHL-F in its light chain, but containing distinct CD28 V domains, a TGN-derived domain in LPD28-10, and a FR104 domain in LPD28-11. In the two-armed form ( Figure 1B (Left), LPD proteins containing different CD28 V domains showed no significant difference in sensitivity to MMP12 cleavage. Figure 5F and 5G ) LPD28-12 and LPD28-13 ( Figure 5HThe LPD light chain (LPD28-13) or LPD heavy chain (LPD28-12) contains LPD connectors LHL and LHL-FR. LHL-FR is designed to be cut by MMP12 relatively faster than LHL-F and LHL LPD connectors. The complete heavy chain in LPD28-12 shows rapid disappearance at 15 min compared to LPD28-10, LPB28-11, and LPD28-13 containing LHL LPD connectors. LPD28-13 retains a significant amount of 75 kDa LB heavy chain strip at the same MMP12 processing time. Comparing the disappearance of complete LPD light chain strips of LPD28-12 and LPD28-13, LPD28-13 with LHL-FR connectors in its LPD light chain disappears faster than LPD28-11 with LHL connectors in its LPD light chain. In LPD28-13-reduced SDS-PAGE, almost no intact LPD light chains were visible after 30 min of MMP12 treatment. In contrast, intact LPD light chains were clearly visible in LPD28-12-reduced SDS-PAGE at 60 min. This confirms that MMP12 increases the sensitivity of LHL-FR linker cleavage in two-armed LPD proteins, regardless of whether the LHL-FR linker is present in the LPD light or heavy chain.

[0178] To assess the ability of undigested, intact proteins and MMP12-incubated proteins to interact with CD28, a binding ELISA of the recombinant human CD28 domain was performed. Simultaneously, purified IgG_001 and IgG_002 proteins were used as positive controls, while isotype binding was used as a negative control. As expected, both IgG_001 and IgG_002 showed strong binding signals to CD28 in the ELISA, while the isotype control showed no binding. Figure 6A Interestingly, all the exemplary proteins tested (LPD28-3, LPD28-10, and LPD-11) were... Figure 6B Both -D) confirmed that CD28 binding was significantly reduced in the intact protein, but strongly increased after incubation with MMP12 at 37°C for a specified time. Specifically, Figure 6B The ELISA results show the binding of undigested (intact / 0 min) or 15 min, 30 min, 1 h, 2 h, 4 h, or 8 h MMP12-incubated protein to human CD28. MMP12-treated LPD28-3 protein bound to CD28 at high levels at all tested concentrations, while undigested LPD28-3 showed a significant CD28 binding signal only at the two highest tested concentrations (12.5 and 100 nM). Figure 6CThe ELISA results show that undigested (0 min) or 15 min, 30 min, 1 h, 2 h, 4 h, or 8 h MMP12-incubated LPD28-10 protein binds to human CD28. MMP12-treated LPD28-10 protein binds to CD28 at all tested concentrations to high levels similar to the bivalent control, while intact LPD28-10 only showed a significant CD28 binding signal at the two highest tested concentrations (12.5 and 100 nM). Figure 6D The ELISA results showed that undigested (0 min) or 15 min, 30 min, 1 h, 2 h, 4 h, or 24 h MMP12-incubated LPD28-11 protein bound to human CD28. At all tested concentrations, MMP12-treated LPD28-11 protein bound to CD28 at levels similar to the bivalent IgG control. Figure 6A Undigested LPD28-11 showed a significant CD28 binding signal only at the three highest concentrations tested (1.5, 12.5, and 100 nM). Figure 6D This data confirms that the binding of undigested / intact LPD28 molecules to recombinant CD28 is severely impaired, but can be restored to full binding affinity through proteolytic activation.

[0179] To further evaluate the ability of undigested, fully intact proteins and MMP12-incubated proteins to interact with CD28, Jurkat cells were subjected to experiments with selected proteins (LPD28-3, LPD28-10, and LPD-11). Figure 7A -C) binding. As previously shown with recombinant proteins, binding signals in Jurkat cells confirmed that in the fully intact LPD protein, binding to the native form of CD28 on the cell surface was significantly reduced. Binding significantly increased after incubation with MMP12 at 37°C for a specified time. Specifically, Figure 7A The binding of intact (0 min) or 15 min, 30 min, 1 h, 2 h, 4 h, or 8 h LPD28-3 protein to Jurkat cells was demonstrated. MMP12-treated LPD28-3 protein bound to Jurkat cell CD28 at high levels at all tested concentrations, while intact LPD28-3 did not show a significant CD28 binding signal at any tested concentration. Figure 7BThe binding of intact (0 min) or 15 min, 30 min, 1 h, 2 h, 4 h, or 8 h LPD28-10 protein to Jurkat cells was demonstrated. MMP12-treated LPD28-10 protein bound to Jurkat cell CD28 at high levels at all tested concentrations, while intact LPD28-10 did not show a significant CD28 binding signal at any tested concentration. Figure 7C The binding of LPD28-11 protein, incubated with MMP12 for 15 min, 30 min, 1 h, 2 h, 4 h, or 24 h, to Jurkat cells was shown. MMP12-treated LPD28-11 protein bound to Jurkat cell CD28 at high levels at all tested concentrations, while undigested LPD28-11 showed only some CD28 binding signal at the two highest tested concentrations (60 and 300 nM). This confirms the relatively high CD28 binding signal of undigested LPD28-11 observed in ELISA. Figure 6D No binding of isotype control cells was detected. Figure 7A -C).

[0180] To demonstrate that the CD28V domain used in the LPD protein is functional and can activate CD28+JurkatIL2 reporter cells as expected, purified IgG protein was tested in this assay. Figure 8The expected activation signal achieved with the control protein is shown. Commercial TGN1412 (IgG4), IgG_001, and IgG_002 were incubated together at specified concentrations with and without CD3 co-stimulation. CD3 co-stimulation was provided by constitutively adding 15 nM anti-CD3 antibody (SP34) to the sample at a specified location. As expected, no luminescent signal was detected in IgG_002 (non-hyperagonist) without CD3 stimulation. In contrast, TGN1412-IgG4 (hyperagonist) showed a significant signal even without CD3 stimulation. IgG_001 contains a V domain highly similar to TGN1412 (IgG4) but is expressed as an IgG1 Fc isotype. IgG_001 showed a significant signal without CD3 stimulation, but less than its TGN1412-IgG4 counterpart. Notably, the CD28 binding capacity of TGN1412 (IgG4) and IgG_001 in ELISA was indistinguishable. The importance and role of Fc isoforms in TGN-mediated CD28 hyperactivation have been well described. This is believed to be primarily related to the spatial differences displayed in the V domain and the relative rigidity of the binding domain determined by the hinge sequences present in the IgG4, IgG2, and IgG1 Fc isoforms (Ball et al., J Immunol 2012). As expected, both TGN1412 and IgG_002 also elicit luminescent signals in the presence of CD3 stimulation.

[0181] In addition, several exemplary LPD proteins with different valence states, LHL linkers, and CD28V domain combinations were selected and their functions were tested in a mixed cell culture assay using the human PD-L1+ cell line MDA-MB-231 bound to the Jurkat cell line, which was engineered to provide a reporter signal for human CD28 activation (IL2 promoter).

[0182] Figure 9A The results show the fold induction of luminescent signals in LPD28-1 and LPD28-2 after 2 h of MMP12 treatment in the presence of CD3 stimulation. LPD28-2 did not show significant signal induction, while LPD28-1 exhibited low signal at the highest concentration, but the activation signal increased with decreasing concentration, followed by a "hook effect" or impaired co-stimulatory capacity at high concentrations. This indicates that CD28 activation is impaired in undigested / intact LPD molecules, but can be restored through protein hydrolysis.

[0183] In addition, the ability of selected LPD proteins to stimulate CD28 on Jurkat IL2 reporter cells was tested under four assay conditions: presence and absence of fixed CD3 stimulation signals, and binding to human PD-L1+ cell line MDA-MB-231 with or without (and thus PDL1 binding).

[0184] Regardless of whether the assay was performed under intact, MMP12-treated, or test conditions, LPD28-3 failed to elicit any CD28 stimulation signal. Figure 9B -E).

[0185] Similarly, regardless of the testing conditions or MMP12 incubation time, the symmetric constructs LPD28-8 and LPD28-9 failed to elicit any CD28 stimulation signal. Figure 9F -M). This indicates that the CD28 V domain, its epitopes, and LPD protein patterns strongly influence its CD28 activation capacity.

[0186] Conversely, two-armed LPD molecules can elicit strong CD28-mediated luminescence responses, depending on the conditions used, the LHL linker combination, and the presence of specific CD28 V domains in the molecule. Figure 10A The results showed that LPD28-10 acquired signal in the presence of CD3 stimulation and MDA-MB-231 cells. Importantly, intact / undigested and 8-h MMP12-treated LPD28-10 showed no signal compared to CD3 stimulation, while treatment with LPD28-10 molecules for 15 min yielded the highest CD28 stimulation signal—accompanied by the pronounced hook effect observed for LPD28-1 above. 30-min MMP12 treatment resulted in a slightly lower CD28 co-stimulatory signal. 1-h and 2-h MMP12-treated LPD28-10 showed significantly higher luminescent signals than CD3 stimulation, confirming the CD28 co-stimulatory capacity of LPD28-10 treated with 15-min, 30-min, 1-h, and 2-h MMP12. In the absence of CD3 co-stimulation but in the presence of MDA-MB-231 cells, intact LPD28-10 did not show a CD28-mediated luminescent signal. Conversely, all MMP12-treated LPD28-10 molecules produced CD28 signals without CD3 stimulation, confirming the ability of the TGN1412-derived CD28 V domain to induce hyperexcitatory signal transduction in the form of two-armed LPD proteins. LPD proteins digested at 15 min and 30 min again showed the highest signal and a pronounced hook effect. LPD28-10 treated at 1 h, 2 h, and 8 h elicited similar luminescent signals, indicating similar levels of digestion and removal of the PDL1-binding domain in LPD28-10. Figure 10B This is consistent with the digestion profile of LPD28-10 in SDS-PAGE. Figure 5FThe absence of MDA-MB-231 cells resulted in the absence of PDL1 binding, leading to a generally higher fold induction rate of Jurkat IL2 reporter cells, indicating that MDA-MB-231 cells possess T-cell suppressive properties. Similarly, intact and 8-h MMP12-digested LPD28-10 proteins showed no stimulatory activity. Conversely, all MMP12-digested LPD proteins exhibited similar luminescent signals, with LPD28-10 treated with MMP12 for 15 min again producing the highest signal. Figure 10C Interestingly, in the absence of PDL1 binding (no MDA-MB-231 cells) and CD3 stimulation, all tested MMP12-treated LPD28-10 molecules stimulated Jurkat IL2 reporter cells to similar degrees. Figure 10D This is related to Figure 10B The image shows a comparison of LPD28-10 fold-induced signal formation in the presence of MDA-MB-231 cells after 15 min and 30 min of treatment. These results indicate the importance of synaptic formation and concurrent PDL1V domain conjugation for conferring optimal (co)stimulatory activity.

[0187] Compared to LPD28-10, the generated LPD28-11 contains the CD28V domain, which cannot generate overexcitation signals. Figure 8 ). Figure 10E The results showed that LPD28-11 could also produce a luminescent signal in the presence of CD3 stimulation and MDA-MB-231 cells. Intact LPD28-11 showed a lower signal at the highest tested concentration (50 nM) compared to CD3 stimulation, confirming the binding results of ELISA and Jurkat cells. Figure 6D and Figure 7C Treatment with LPD28-11 molecules for 15 and 30 min yielded the highest CD28 stimulation signals—again accompanied by the pronounced hook effect observed above in LPD28-1 and LPD28-10. MMP12-treated LPD28-11 at 1 h and 2 h showed significantly higher luminescence signals than CD3-stimulated LPD28-11, confirming the CD28 co-stimulatory capacity of MMP12-treated LPD28-11 at 15 min, 30 min, 1 h, and 2 h. In the absence of CD3 co-stimulation but with the presence of MDA-MB-231 cells, as expected, intact or MMP12-treated LPD28-11 did not show CD28-mediated luminescence signals. Figure 10FAs mentioned above, due to the absence of MDA-MB-231 cells, PDL1 binding was absent, and the overall fold induction of Jurkat IL2 reporter cells was again higher in the presence of CD3 stimulation. Again, intact digested LPD28-11 showed stimulatory activity at the highest concentration tested. Figure 10G In contrast, all MMP12-digested LPD proteins exhibited sustained stimulatory activity at three of the five tested concentrations and achieved similar fold-up induction across all MMP12 treatment durations. Figure 10G As expected, in the absence of PDL1 binding (no MDA-MB-231 cells) and CD3 stimulation, neither the intact nor MMP12-treated LPD28-11 molecules tested stimulated Jurkat IL2 reporter cells. Figure 10H ).

[0188] Other two-armed LPD molecules containing the TGN1412-derived CD28-binding V domain were tested in a similar manner: LPD28-12 and LPD28-13. Both LPD28-12 and LPD28-13 contain an LHL-FR connector, designed to impart faster MMP12 cutting capability, which in... Figure 5H The results for LPD28-12 were confirmed, but those for LPD28-13 were not confirmed. Figure 10I The results showed that LPD28-12 acquired a signal in the presence of CD3 stimulation and MDA-MB-231 cells. Importantly, intact LPD28-12 showed the least signal compared to CD3 stimulation at the highest concentration tested. Treatment with LPD28-12 molecules for 15 min and 30 min, respectively, yielded strong CD28 stimulation signals—again accompanied by the prominent hook effect observed for LPD proteins previously. MMP12-treated LPD28-12 at 1 h, 2 h, and 24 h showed similarly significantly higher luminescent signals than CD3 stimulation, confirming the CD28 co-stimulatory capacity of MMP12-treated LPD28-12 at 15 min, 30 min, 1 h, 2 h, and 24 h. Figure 10I In the absence of CD3 co-stimulation but in the presence of MDA-MB-231 cells, intact LPD28-12 cells did not exhibit CD28-mediated luminescence. Figure 10J Conversely, all MMP12-treated LPD28-12 molecules generated CD28 signals without requiring CD3 stimulation, confirming the ability of the TGN1412-derived CD28 V domain to induce hyperexcitatory signal transduction as a two-armed LPD protein. The degree of co-stimulation induced by LPD28-12 did not change significantly over MMP12 treatment time, consistent with the digestion profile of LPD28-12 in SDS-PAGE. Figure 5H This indicates that the intact heavy chain was completely digested at 15 min, while some remaining intact light chains were retained until the 30 min digestion time point. The absence of MDA-MB-231 cells, and therefore the lack of PDL1 binding, again resulted in a higher overall fold increase in induction of Jurkat IL2 reporter cells. Furthermore, intact LPD28-12 protein showed some stimulatory activity at the highest concentration tested. Figure 10K In contrast, all MMP12-digested LPD proteins showed similar luminescent signals, with LPD28-12 treatment for 15 min resulting in the strongest signal. Figure 10K As observed with LPD28-10, in the absence of PDL1 binding (no MDA-MB-231 cells) and CD3 stimulation, all tested MMP12-treated LPD28-12 molecules stimulated Jurkat IL2 reporter cells to an almost indistinguishable degree. Figure 10L ).

[0189] Next, LPD28-13 was tested using Jurkat IL2 reporter cells as described. Figure 10M The results showed that LPD28-13 acquired a signal in the presence of CD3 stimulation and MDA-MB-231 cells. Importantly, intact LPD28-13 did not show a signal compared to CD3 stimulation. Treatment with LPD28-13 molecules for 30 min and 1 h yielded a strong CD28 stimulation signal—accompanied by a pronounced hook effect as previously observed with LPD proteins. LPD28-13 treated with MMP12 at 15 min, 2 h, and 24 h showed a similarly significantly higher luminescent signal than CD3 stimulation, confirming the CD28 co-stimulatory capacity of LPD28-12 treated with MMP12 at 15 min, 30 min, 1 h, 2 h, and 24 h as described above. Figure 10M In the absence of CD3 co-stimulation but in the presence of MDA-MB-231 cells, intact LPD28-13 cells did not exhibit CD28-mediated luminescence. Figure 10N Conversely, all MMP12-treated LPD28-13 molecules produced CD28 signaling without CD3 stimulation. Peak levels were reached at the 30-minute digestion time point. All remaining MMP12-treated LPD28-13 proteins showed lower levels compared to background-induced co-stimulation. Figure 10N The absence of MDA-MB-231 cells, and therefore the lack of PDL1 binding, again resulted in a higher overall fold increase in induction of Jurkat IL2 reporter cells. The intact LPD28-13 protein showed some stimulatory activity at its highest tested concentration. Figure 10OAll MMP12-digested LPD proteins showed similar luminescent signals. Figure 10O As observed with LPD28-10, the results in the absence of PDL1 binding (no MDA-MB-231 cells) and CD3 stimulation were compared to those in the presence of MDA-MB-231 cells. Figure 10M And N), all tested MMP12-treated LPD28-13 molecules stimulated Jurkat IL2 reporter cells to very similar degrees. Figure 10L In summary, these data confirm the importance of synaptic formation and concurrent PDL1 V domain conjugation for optimal (co-)stimulatory CD28 activity conferred by the LPD protein. This indicates that the CD28 V domain, its epitopes, LPD adaptor design, and LPD protein pattern strongly influence the LPD protein's ability to activate CD28.

[0190] To further examine the effect of LPD proteins on stimulating immune activation, representative LPD28 protein was tested in a mixed lymphocyte reaction assay. Briefly, monocytes isolated from a donor's peripheral blood mononuclear cells (PBMCs) were differentiated into immature dendritic cells (iDCs) and then co-cultured with CD3+ T cells from an unmatched donor. This was to generate low levels of CD3 activation signaling in T cells. MDA-MB-231 cells were incubated with the mixed lymphocyte reaction in the presence or absence of intact or MMP12-treated LPD molecules. Test and / or control compounds were added, and the production of the cytokines interleukin-2 (IL-2) and interferon-γ (IFN-γ) was measured by ELISA after two and five days, respectively, to assess the immunomodulatory potential of the selected LPD protein. In mixed lymphocyte reactions, intact and 2-h MMP12-treated LPD28-1 were incubated at indicated concentrations C1 = 20 nM, C2 = 6.6 nM, and C3 = 2.2 nM in the presence or absence of MDA-MB-231 cells (Figs. 11A1, 11A2, and B). The presence of IL-2 or IFNg in the supernatant was used as a biomarker for T cell activity. Neither intact nor 2-h MMP12-treated LPD28-1 protein resulted in significant IL-2 or IFNg release compared to background MLR signaling in the presence or absence of MDA-MB-231 cells. Three donor pairs were tested, and results for representative donor pairs are shown in Figs. 11A1, 11A2, and B. Conversely, LPD28-10 MMP12 treatment induced strong T cell activity, resulting in significantly higher levels of IL2 and IFNg secretion than background MLR levels and atezolizumab (PDL1 blockade only) at all tested concentrations and donor combinations (C1 = 20 nM, C2 = 7 nM, and C3 = 2 nM) (Fig. 11C1-3 and D1-3, Fig. 11E1-3 and Fig. 11F1-3). The lowest LPD28-10 concentrations showed the highest cytokine secretion, again demonstrating the hook effect observed in Jurkat IL2 reporter cells. Importantly, intact LPD28-10 protein did not induce cytokine secretion above control PDL1 blockade levels (ezolizumab control) (IL2: Fig. 11C1-3 and D1-3; IFNg: Fig. 11E1-3 and Fig. 11F1-3). This supports the idea that LPD design and patterns can strongly influence its ability to induce cytokine release in primary cells.

[0191] To examine the effect of combining PD-L1 blockade with potent CD28 co-stimulation, selected LPD proteins were tested using primary T cells to assess their potential to mediate cytotoxic killing in MDA-MD-231 cells. This effect was measured by inducing annexin signaling in MDA-MB-231 cells. Figure 12 First, baseline cytotoxicity under buffer conditions was established using only commercially available TGN1412 (IgG4) and TGN-derived CD28 V domain IgG_001 (against the IgG1-Fc isotype). Figure 12 Within hours of adding TGN1412 (IgG4), cytotoxicity levels were significantly higher than background levels, and for IgG_001, cytotoxicity levels were also significantly higher than background levels approximately 48 hours after compound addition. This further confirms the tunable nature of hyperactivation and the higher activity of hyperactivating antibodies with the IgG4 isotype.

[0192] LPD28-1, LPD28-10, and LPD28-12 proteins were incubated on the same plate and compared with a PDL1xCD3 conjugate as a positive control. No CD3 co-stimulation was provided. Undigested LPD28-1 showed low levels of cytotoxicity compared to background, but significantly lower than the PDL1xCD3 positive control. MMP12 treatment (2 h) of LPD28-1 showed increased cytotoxicity compared to the undigested molecule, but again lower than the positive control. Figure 13A This cytotoxic activity is surprising because CD28-mediated cytotoxicity without CD3 co-stimulation has not been described before. Interestingly, no cytokine secretion was detected in the MLR assay of LPD28-1 (Figure 11), which may indicate that the compound has an interesting phenotype of both conferring cytotoxicity and exhibiting low concomitant cytokine release. Similarly, intact LPD28-10, LPD28-12, and LPD28-13 showed low levels of cytotoxicity after treatment with MMP12 at specified time points, while annexin signaling was significantly increased. Figure 13B LPD28-10 molecules were tested after 30 min and 2 h MMP12 treatment. Interestingly, LPD28-10 treated with 2 h MMP12 induced annexin signaling (a measure of cytotoxicity) less than LPD28-10 treated with 30 min MMP12. Figure 13B Prolonged MMP12 treatment resulted in a higher degree of complete molecular cleavage and PDL1 Fab removal. This again demonstrates the need for simultaneous PDL-1 and CD28 conjugation to optimally activate T cells and guide activity. Furthermore, the compounds treated with 1 nM and 10 nM MMP12 were used to titrate LPD28-12 and LPD28-13 (…). Figure 13C This leads to higher overall cytotoxicity at lower concentrations (InM) – again demonstrating that the hook effect at higher compound concentrations results in impaired immune cell activity. In summary, combining PDL1 blockade with strong CD28 stimulation in LPD molecules enables single-agent cytotoxic activity without CD3 co-stimulation.

[0193] To examine the safety potential of selected intact / undigested LPD compounds, a cytokine release assay was performed. Briefly, all proteins were coated at a high density (1 μg / ml) onto 96-well plates and incubated with PBMCs from 20 different healthy donors for 72 h. The supernatant was then harvested and cytokine secretion was measured. Figure 13A -F). As expected, high levels of IL2 secretion were detected in both TGN1412 (IgG4) and IgG_001 (a CD28 binder on the IgG1 Fc backbone derived from the TGN1412 V domain)—the latter stimulating lower IL2 release. This further confirms the previously observed and anticipated quality of IgG4 and IgG1 isoforms in CD28 hyperactivation. IL2 secretion levels from TGN1412 (IgG4) were consistently high in most donors, resulting in IL2 levels as high as 80,000 pg / ml—the upper limit of quantitation measured, comparable to the anti-CD3 (OKT3) positive control. IgG_001 elicited an average IL2 secretion of approximately 2,000 pg / ml in PBMCs from 20 healthy donors tested. The two-arm LPD molecules LPD28-10, LPD28-12, and LPD28-13 did not result in a mean IL2 secretion exceeding 100 pg / ml, similar to the average levels achieved in PBS, Erbitux, and atezolizumab tested as negative controls in the same donors. This result confirms that the intact LPD molecules effectively protect the CD28V domain from its interaction with its target on CD28+ T cells. Additional single-arm LPD molecules LPD 28-4 and LPD 28-5 were also tested in this assay, showing similar low levels of mean IL2 secretion (<100 pg / ml) as described for the two-arm LPD molecules (5 donors tested in total). Figure 14A ).

[0194] Next, the IFNg secretion levels of TGN1412 (IgG4) and IgG_001 (a CD28 binder derived from the TGN1412 V domain on the IgG1 Fc backbone) were measured in the same supernatant. The latter stimulated lower IFNg release, further confirming the previously observed and expected quality of the IgG4 and IgG1 backbone's hyperagonistic effect on CD28. IFNg secretion levels from TGN1412 (IgG4) were consistently high in most donors, resulting in IFNg levels as high as 20,000 pg / ml—the upper limit of quantitation measured, comparable to the anti-CD3 (OKT3) positive control. IgG_001 induced an average IFNg secretion of approximately 800 pg / ml in the supernatant. Surprisingly, the two-arm LPD molecules LPD28-10, LPD28-12, and LPD28-13 did not result in a mean IFNg secretion exceeding 200 pg / ml, similar to the average levels achieved in PBS, cetuximab, and atezolizumab as negative controls tested in the same donor. The other single-arm LPD molecules, LPD 28-4 and LPD 28-5, were also tested in this assay, and these compounds showed similar low levels of mean IFNg secretion (<100 pg / ml) as described for the two-arm LPD molecules (5 donors tested in total). Figure 14B ).

[0195] Next, IL-10 secretion levels of TGN1412 (IgG4) and IgG_001 (a CD28 binder derived from the TGN1412 V domain on the IgG1 Fc backbone) were measured in the same supernatant. The latter stimulated lower IL-10 release, further confirming the previously observed and expected quality of the IgG4 and IgG1 backbone's hyperagonistic effect on CD28. IL-10 secretion levels of TGN1412 (IgG4) from most donors were consistently high, resulting in an average IL-10 level of approximately 5000 pg / ml, lower than the average level (approximately 20000 pg / ml) achieved in the anti-CD3 (OKT3) positive control. IgG_001 induced an average IL-10 secretion of approximately 2000 pg / ml in the supernatant. Surprisingly, the two-arm LPD molecules LPD28-10, LPD28-12, and LPD28-13 did not result in a mean IL-10 secretion exceeding 100 pg / ml, similar to the average levels achieved in PBS, cetuximab, and atezolizumab as negative controls tested in the same donor. Additional single-arm LPD molecules LPD 28-4 and LPD 28-5 were also tested in this assay, and these compounds showed similar low levels of mean IL-10 secretion (<100 pg / ml) as described for the two-arm LPD molecules (5 donors tested in total). Figure 14C ).

[0196] Next, IL-13 secretion levels of TGN1412 (IgG4) and IgG_001 (a CD28 binder derived from the TGN1412 V domain on the IgG1 Fc backbone) were measured in the same supernatant. The latter stimulated lower IL-13 release, further confirming the previously observed and expected quality of the IgG4 and IgG1 backbone's CD28 hyperagonistic effect. IL-13 secretion levels of TGN1412 (IgG4) from most donors were consistently high, resulting in an average IL-13 level of approximately 8000 pg / ml, significantly higher than the average level (approximately 1000 pg / ml) achieved in the anti-CD3 (OKT3) positive control. IgG_001 induced an average IL-13 secretion of approximately 5000 pg / ml in the supernatant. Surprisingly, the mean IL-13 level of the two-arm LPD molecule LPD28-10 did not exceed 100 pg / ml, similar to the near-average levels achieved in PBS, cetuximab, and atezolizumab as negative controls tested in the same donor. The mean levels of LPD28-12 and LPD28-13 reached approximately 100 pg / ml. The other single-arm LPD molecules, LPD 28-4 and LPD 28-5, were also tested in this assay, and these compounds showed similarly low mean IL-10 secretion levels (<100 pg / ml) as described for the two-arm LPD molecules (5 donors tested in total). Figure 14D ).

[0197] Next, TNFα secretion levels of TGN1412 (IgG4) and IgG_001 (a CD28 binder derived from the TGN1412 V domain on the IgG1 Fc isotype) were measured in the same supernatant. The latter stimulated lower TNFα release, further confirming the previously observed and expected quality of the IgG4 and IgG1 backbone's hyperagonistic effect on CD28. Most donors showed consistently high TNFα secretion levels of TGN1412 (IgG4), resulting in an average TNFα level of approximately 30,000 pg / ml—comparable to the average level achieved in the anti-CD3 (OKT3) positive control. IgG_001 induced an average TNFα secretion of approximately 10,000 pg / ml in the supernatant. Surprisingly and interestingly, the two-arm LPD molecules LPD28-10, LPD28-12, and LPD28-13 did not result in a mean TNFα secretion exceeding 350 pg / ml, similar to the average levels achieved in PBS, cetuximab, and atezolizumab as negative controls tested in the same donor. The other single-arm LPD molecules, LPD 28-4 and LPD28-5, were also tested in this assay, and these compounds showed similar low levels of mean TNFα secretion (<100 pg / ml) as described for the two-arm LPD molecules (5 donors tested in total). Figure 14E ).

[0198] Finally, IL-6 secretion levels of TGN1412 (IgG4) and IgG_001 (a CD28 binder derived from the TGN1412 V domain on the IgG1 Fc isotype) were measured in the same supernatant—the latter stimulating lower IL-6 release, further confirming the previously observed and anticipated quality of the IgG4 and IgG1 backbone's CD28 hyperactivation. TGN1412 (IgG4) IL-6 secretion levels were consistently high in most donors, resulting in an average IL-6 level of approximately 20,000 pg / ml (the upper limit of the LOQ in this assay)—comparable to the average level achieved in the anti-CD3 (OKT3) positive control. IgG_001 induced an average IL-6 secretion of approximately 300 pg / ml in the supernatant. The two-arm LPD molecules LPD28-10, LPD28-12, and LPD28-13 did not result in a mean IL-6 secretion exceeding 200 pg / ml, similar to the average levels achieved in PBS, cetuximab, and atezolizumab as negative controls tested in the same donor. Additional single-arm LPD molecules LPD 28-4 and LPD 28-5 were also tested in this assay, and these compounds showed similar low levels of mean IL-6 secretion (<100 pg / ml) as described for the two-arm LPD molecules (5 donors tested in total). Figure 14FCompared to unmasked CD28 activators, LPD molecules show a significantly increased safety potential.

[0199] Next, to examine the safety potential of the selected intact / undigested LPD compounds, we attempted to test the selected LPD molecules in two additional cytokine release assays. Briefly, soluble and intact LPD28-1 and LPD28-2 molecules (C1 = 15 μg / mL, C2 = 2 μg / mL, and C3 = 1 μg / mL) were incubated with specified concentrations of PBMCs isolated from 10 healthy donors. Cytokine secretion from the supernatant was measured after 24 h. Figure 15A ). Figure 15A The mean fold change compared to the buffer control is shown. Interestingly, TGN1412 IgG4 was able to induce a strong fold change in the release of the tested cytokines (IL-2, IL-6, IL-8, IL-10, IL-13, IFN-γ, and TNF-α), while IgG_001 did not produce a similar level of change in cytokine secretion. Importantly, neither LPB28-1 nor LPB28-2 showed a significant fold change compared to background for any of the tested cytokines.

[0200] Next, the compounds were tested using a third PBMC cytokine release assay (called the RESTORE assay). This assay was first described and tested by Romer et al. in Immunobiology 2011, attempting to establish a reliable PBMC assay for the detection of hyperagonistic effects. Briefly, PBMCs were isolated from 10 healthy donors as before. However, unlike the conventional PBMC cytokine release assay, the PBMCs were incubated at 37°C for 48 h. After the incubation period, a soluble control compound, along with intact LPD28-1 and LPD28-2 molecules (C1 = 15 μg / mL, C2 = 2 μg / mL, and C3 = 1 μg / mL), was mixed with the PBMCs that had been left to stand for 48 h. Then, as before, cytokine secretion from the supernatant was detected after 24 h. Figure 15B ). Figure 15BThe results showed a mean fold change compared to the buffer control. As before, TGN1412 IgG4 elicited strong cytokine release of the following tested cytokines: IL-2, IL-6, IL-8, IL-10, IL-13, IFN-γ, and TNF-α. However, this time IgG_001 also produced a significant change in cytokine secretion, confirming the increased sensitivity of this assay to compounds with CD28 hyperactivating properties. Importantly, in the RESTORE assay, neither LPB28-1 nor LPB28-2 showed a significant fold change from background for any of the tested cytokines. This confirms that intact LPB28-1 and LPB28-2 cannot stimulate cytokine secretion in the PBMC safety assay.

[0201] Implementation Plan 1. A protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, The heavy chain comprises, in order from N-end to C-end, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first joint, an anti-CD28 VH domain, and a second CH1 domain; The light chain comprises, in order 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-CD28 VL domain, and a second immunoglobulin light chain constant region.

[0202] 2. The protein according to embodiment 1, 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-CD28 VH domain, the second CH1 domain, the hinge, the CH2 domain, and the CH3 domain.

[0203] 3. The protein according to embodiment 1 or 2, wherein the protein further comprises a third polypeptide chain, the third polypeptide chain comprising a hinge and an Fc region.

[0204] 4. The protein according to embodiment 3, wherein the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:32.

[0205] 5. The protein according to any one of embodiments 1-4, wherein the first linker comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0206] 6. The protein according to any one of embodiments 1-5, wherein the second linker comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0207] 7. The protein according to any one of embodiments 1-6, The anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:12, HCDR2 containing the amino acid sequence of SEQ ID NO:13, and HCDR3 containing the amino acid sequence of SEQ ID NO:14. The anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:15, LCDR2 containing the amino acid sequence of SEQ ID NO:16, and LCDR3 containing the amino acid sequence of SEQ ID NO:17. The anti-CD28 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:18, HCDR2 containing the amino acid sequence of SEQ ID NO:19, and HCDR3 containing the amino acid sequence of SEQ ID NO:20. The anti-CD28 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:21, LCDR2 containing the amino acid sequence of SEQ ID NO:22, and LCDR3 containing the amino acid sequence of SEQ ID NO:23.

[0208] 8. The protein according to any one of embodiments 1-7, wherein the anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO:24, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO:25.

[0209] 9. The protein according to any one of embodiments 1-8, wherein the anti-CD28 VH domain comprises the amino acid sequence of SEQ ID NO:26.

[0210] 10. The protein according to any one of embodiments 1-9, wherein the anti-CD28 VL domain comprises the amino acid sequence of SEQ ID NO:27.

[0211] 11. The protein according to any one of embodiments 1-10, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:31.

[0212] 12. The protein according to any one of embodiments 1-11, wherein the light chain comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:29.

[0213] 13. The protein according to embodiment 3, wherein (a) The heavy chain comprises the amino acid sequence of SEQ ID NO:30, the light chain comprises the amino acid sequence of SEQ ID NO:28, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:32; or (b) The heavy chain contains the amino acid sequence of SEQ ID NO:31, the light chain contains the amino acid sequence of SEQ ID NO:29, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO:32.

[0214] 14. The protein according to embodiment 2 or 3, wherein the heavy chain comprises a constant region of IgG, IgE, IgM, IgD, IgA or IgY.

[0215] 15. The protein according to embodiment 2 or 3, wherein the heavy chain comprises a constant region of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0216] 16. The protein according to embodiment 2 or 3, wherein the heavy chain comprises an immune inert constant region.

[0217] 17. The protein according to embodiment 2 or 3, wherein the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region comprising 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 amino acid substitution S228P, wherein the numbering is based on the EU index as in Kabat.

[0218] 18. An immunoconjugate comprising a protein according to any one of embodiments 1-17 linked to a therapeutic agent.

[0219] 19. The immunoconjugate according to embodiment 18, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell inhibitory enzyme, a cell lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or an apoptosis-promoting agent.

[0220] 20. A pharmaceutical composition comprising a protein according to any one of embodiments 1-17 or an immunoconjugate according to embodiment 18 or 19, and a pharmaceutically acceptable carrier, diluent or excipient.

[0221] 21. A nucleic acid molecule encoding a protein according to any one of embodiments 1-17. (a) Heavy chain amino acid sequence; (b) Light chain amino acid sequence; or (c) Both the heavy chain amino acid sequence and the light chain amino acid sequence.

[0222] 22. An expression vector comprising a nucleic acid molecule according to embodiment 21.

[0223] 23. A recombinant host cell comprising a nucleic acid molecule according to embodiment 21 or an expression vector according to embodiment 22.

[0224] 24. A method for producing a protein, the method comprising: Recombinant host cells containing the expression vector according to embodiment 22 are cultured under conditions where the nucleic acid molecule can be expressed, thereby producing proteins; and The protein is isolated from the host cell or culture.

[0225] 25. A method for enhancing an anticancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of embodiments 1-17, an immunoconjugate according to embodiment 18 or 19, or a pharmaceutical composition according to embodiment 20.

[0226] 26. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of embodiments 1-17, an immunoconjugate according to embodiment 18 or 19, or a pharmaceutical composition according to embodiment 20.

[0227] 27. A method for alleviating cancer symptoms in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of embodiments 1-17, an immunoconjugate according to embodiment 18 or 19, or a pharmaceutical composition according to embodiment 20.

[0228] 28. The method according to any one of embodiments 25-27, wherein the cancer is gastrointestinal stromal carcinoma (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 bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy.

[0229] 29. A protein comprising a first heavy chain and a first light chain. The first heavy chain includes: (a) A first portion (P1) of the first antigen-binding domain, comprising a first heavy chain variable domain (VH) and an optional first heavy chain constant domain 1 (CH1); (b) a first adapter, wherein the first adapter is cleavable by a protease; and (c) The first portion (P2) of the second antigen-binding domain, comprising the second VH and optionally the second CH1; wherein: (i) The N-end of the first connector is connected to the C-end of the first CH1 of P1, and the C-end of the first connector is connected to the N-end of the second VH of P2; or (ii) The N-terminal of the first connector is connected to the C-terminal of the first VH of P1, and the C-terminal of the first connector is connected to the N-terminal of the second VH of P2; and The first light chain comprises: (a) The second portion (P3) of the first antigen-binding domain includes a first light chain variable domain (VL) and an optional first light chain constant domain (CL); (b) a second connector, wherein the second connector is cleavable by a protease; and (d) A second portion (P4) of the second antigen-binding domain, comprising a second VL and an optional second CL; wherein: (i) The N-end of the second connector is connected to the C-end of the first CL of P3, and the C-end of the second connector is connected to the N-end of the second VL of P4; or (ii) The N-end of the first connector is connected to the C-end of the first VL of P3, and the C-end of the second connector is connected to the N-end of the second VL of P4; and Wherein the first antigen-binding domain binds to PD-L1; and When the first and second connectors are cleaved, the second antigen-binding domain binds to CD28.

[0230] 30. The protein according to embodiment 29, wherein the CH1 of P1 is covalently linked to the CL of P3.

[0231] 31. The protein according to embodiment 29 or 30, wherein the CH1 of P2 is covalently linked to the CL of P4.

[0232] 32. A protein comprising a first heavy chain and a first light chain. The first heavy chain includes: (a) A first portion (P1) of the first antigen-binding domain, which includes a first heavy chain variable domain (VH); (b) a first adapter, wherein the first adapter is cleavable by a protease; and (c) The first portion (P2) of the second antigen-binding domain, which contains the second VH; The N end of the first connector is connected to the C end of P1, and the C end of the first connector is connected to the N end of the second VH of P2. The first light chain comprises: (c) The second portion (P3) of the first antigen-binding domain includes a first light chain variable domain (VL); (d) a second connector, wherein the second connector is cleavable by a protease; and (d) The second portion (P4) of the second antigen-binding domain, which includes the second VL; The N end of the second connector is connected to the C end of P3, and the C end of the second connector is connected to the N end of the second VL of P4. Wherein the first antigen-binding domain binds to PD-L1; and When the first and second connectors are cleaved, the second antigen-binding domain binds to CD28.

[0233] 33. The protein according to embodiment 32, wherein P1 further comprises a first heavy chain constant domain 1 (CH1), wherein the C-terminus of the first CH1 of P1 is connected to the N-terminus of the first linker.

[0234] 34. The protein according to embodiment 33, wherein the N-terminus of the first CH1 of P1 is connected to the C-terminus of the VH of P1.

[0235] 35. The protein according to embodiment 32, wherein the P3 further comprises a first CL, wherein the C-terminus of the first CL of the P3 is connected to the N-terminus of the second connector.

[0236] 36. The protein according to embodiment 35, wherein the N-terminus of the first CL of P3 is connected to the C-terminus of the first VL of P3.

[0237] 37. The protein according to any one of embodiments 32-36, wherein the P2 further comprises a second CH1, wherein the N-terminus of the second CH1 of P2 is connected to the C-terminus of the second VH of P2.

[0238] 38. The protein according to any one of embodiments 32-37, wherein the P4 further comprises a second CL, wherein the N-terminus of the second CL of the P4 is connected to the C-terminus of the second VL of the P4.

[0239] 39. The protein according to any one of embodiments 35-38, wherein the first CH1 of P1 is covalently linked to the first CL of P3.

[0240] 40. The protein according to any one of embodiments 35-39, wherein the second CH1 of P2 is covalently linked to the second CL of P4.

[0241] 41. The protein according to any one of embodiments 29-40, wherein the first antigen-binding domain comprises P1 and P3.

[0242] 42. The protein according to any one of embodiments 29-41, wherein P1 and P3 are covalently linked to form Fab.

[0243] 43. The protein according to any one of embodiments 29-42, wherein the second antigen-binding domain comprises P2 and P4.

[0244] 44. The protein according to any one of embodiments 29-43, wherein P2 and P4 are covalently linked to form Fab.

[0245] 45. The protein according to any one of embodiments 29-44, wherein the first heavy chain further comprises a first dimerizing domain.

[0246] 46. ​​The protein according to embodiment 45, wherein the first dimerizing domain comprises a first hinge domain and a first immunoglobulin constant domain (Fc domain).

[0247] 47. The protein according to embodiment 46, wherein the Fc domain comprises a first heavy chain constant domain 2 (CH2) and a first heavy chain constant domain 3 (CH3).

[0248] 48. The protein according to embodiments 45-47, wherein the C-terminus of P2 is connected to the N-terminus of the first hinge domain.

[0249] 49. The protein according to any one of embodiments 29-48, wherein the first linker comprises an amino acid sequence having at least 85% identity with any one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:33.

[0250] 50. The protein according to embodiment 49, wherein the first linker comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:33.

[0251] 51. The protein according to any one of embodiments 29-50, wherein the second linker comprises an amino acid sequence having at least 85% identity with any one of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:33.

[0252] 52. The protein according to embodiment 51, wherein the second linker comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:33.

[0253] 53. The protein according to any one of embodiments 29-52, wherein the first VH of P1 comprises: (a) The amino acid sequence of the heavy chain complementarity-determining region 1 (HCDR1) of SEQ ID NO:12, SEQ ID NO:34 or SEQ ID NO:36; (b) The amino acid sequence of the heavy chain complementarity-determining region 2 (HCDR2) of SEQ ID NO:13, SEQ ID NO:35, or SEQ ID NO:37; and (c) The amino acid sequence of the heavy chain complementarity-determining region 3 (HCDR3) of SEQ ID NO:14 or SEQ ID NO:38.

[0254] 54. The protein according to any one of embodiments 29-53, wherein the first VL of P3 comprises: (a) The amino acid sequence of the light chain complementarity-determining region 1 (LCDR1) of SEQ ID NO:15 or SEQ ID NO:39; (b) a light chain variable domain comprising the amino acid sequence of light chain complementarity-determining region 1 (LCDR2) of SEQ ID NO:16 or DA; and (c) Light chain variable domain, which contains the amino acid sequence of light chain complementarity determination region 3 (LCDR3) of SEQ ID NO:17.

[0255] 55. The protein according to any one of embodiments 29-54, wherein the second VH of P2 comprises: (a) The HCDR1 amino acid sequence of SEQ ID NO:40, SEQ ID NO:43 or SEQ ID NO:45; (b) The HCDR2 amino acid sequence of SEQ ID NO:41, SEQ ID NO:44, or SEQ ID NO:46; and (c) The HCDR3 amino acid sequence of SEQ ID NO:42 or SEQ ID NO:47.

[0256] 56. The protein according to any one of embodiments 29-55, wherein the second VL of P4 comprises: (a) The LCDR1 amino acid sequence of SEQ ID NO:48 or SEQ ID NO:51; (b) The LCDR2 amino acid sequence or KA of SEQ ID NO:49; and (c) The LCDR3 amino acid sequence of SEQ ID NO:50.

[0257] 57. The protein according to any one of embodiments 29-54, wherein the second VH of P2 comprises: (a) The HCDR1 amino acid sequence of SEQ ID NO:53, SEQ ID NO:56 or SEQ ID NO:58; (b) The HCDR2 amino acid sequence of SEQ ID NO:54, SEQ ID NO:57, or SEQ ID NO:59; and (c) The HCDR3 amino acid sequence of SEQ ID NO:55 or SEQ ID NO:60.

[0258] 58. The protein according to any one of embodiments 29-54 and 57, wherein the second VL of P4 comprises: (a) The LCDR1 amino acid sequence of SEQ ID NO:61 or SEQ ID NO:64; (b) The LCDR2 amino acid sequence or AA of SEQ ID NO:62; and (c) The LCDR3 amino acid sequence of SEQ ID NO:63.

[0259] 59. The protein according to any one of embodiments 29-58, wherein the first VH of P1 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:24.

[0260] 60. The protein according to any one of embodiments 29-59, wherein the first VH of P1 comprises the sequence of SEQ ID NO:24.

[0261] 61. The protein according to any one of embodiments 29-60, wherein the first VL of P3 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:25.

[0262] 62. The protein according to any one of embodiments 29-61, wherein the first VL of P3 comprises the sequence of SEQ ID NO:25.

[0263] 63. The protein according to any one of embodiments 29-56 and 59-62, wherein the second VH of P2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:26.

[0264] 64. The protein according to any one of embodiments 29-56 and 59-63, wherein the second VH of P2 comprises the sequence of SEQ ID NO:26.

[0265] 65. The protein according to any one of embodiments 29-56 and 59-64, wherein the second VL of P4 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:27.

[0266] 66. The protein according to any one of embodiments 29-56 and 59-65, wherein the second VL of P4 comprises the sequence of SEQ ID NO:27.

[0267] 67. The protein according to any one of embodiments 29-54 and 58-62, wherein the second VH of P3 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:66.

[0268] 68. The protein according to any one of embodiments 29-54, 58-62 and 67, wherein the second VH of P3 comprises the sequence of SEQ ID NO:66.

[0269] 69. The protein according to any one of embodiments 29-54, 58-62, 67 and 68, wherein the second VL of P4 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:67.

[0270] 70. The protein according to any one of embodiments 29-54, 58-62 and 67-69, wherein the second VL of P4 comprises the sequence of SEQ ID NO:67.

[0271] 71. The protein according to any one of embodiments 29-56 and 59-66, wherein the first heavy chain comprises: (a) The first VH of P1 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:24; and (b) The second VH of P2 has at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:26.

[0272] 72. The protein according to any one of embodiments 29-56, 59-66, and 71, wherein the first heavy chain comprises: (a) The first VH of P1, which contains the sequence of SEQ ID NO:24; and (b) The second VH of P2, which contains the sequence of SEQ ID NO:26.

[0273] 73. The protein according to any one of embodiments 29-54, 58-62, and 67-70, wherein the first heavy chain comprises: (a) The first VH of P1 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:24; and (b) The second VH of P2 has at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:66.

[0274] 74. The protein according to any one of embodiments 29-54, 58-62, 67-70, and 73, wherein the first heavy chain comprises: (a) The first VH of P1, which contains the sequence of SEQ ID NO:24; and (b) The second VH of P2, which contains the sequence of SEQ ID NO:66.

[0275] 75. The protein according to any one of embodiments 29-74, wherein the first light chain comprises: (a) The first VL of P3, which has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:25; and (b) The second VL of P4 has at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:27.

[0276] 76. The protein according to any one of embodiments 29-75, wherein the first light chain comprises: (a) The first VL of P3, which contains the sequence of SEQ ID NO:25; and (b) The second VL of P4, which contains the sequence of SEQ ID NO:27.

[0277] 77. The protein according to any one of embodiments 29-74, wherein the first light chain comprises: (a) The first VL of P3, which has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:25; and (b) The second VL of P4, which has at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:67.

[0278] 78. The protein according to any one of embodiments 29-75, wherein the first light chain comprises: (a) The first VL of P3, which contains the sequence of SEQ ID NO:25; and (b) The second VL of P4, which contains the sequence of SEQ ID NO:67.

[0279] 79. The protein according to any one of embodiments 29-78, (a) wherein the first heavy chain comprises: (i) The first VH of P1 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:24; and (ii) The second VH of P2, which has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:26; and (b) wherein the first light chain comprises: (i) The first VL of P3 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:25; and (ii) The second VL of P4, which has at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:27.

[0280] 80. The protein according to any one of embodiments 29-79, (a) wherein the first heavy chain comprises: (i) The first VH of P1, which contains the sequence of SEQ ID NO:24; and (ii) The second VH of P2, which contains the sequence of SEQ ID NO:26; and (b) wherein the first light chain comprises: (i) The first VL of P3, which contains the sequence of SEQ ID NO:25; and (ii) The second VL of P4, which contains the sequence of SEQ ID NO:27.

[0281] 81. The protein according to any one of embodiments 29-78, (a) wherein the first heavy chain comprises: (i) The first VH of P1 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:24; and (ii) The second VH of P2, which has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:66; and (b) wherein the first light chain comprises: (i) The first VL of P3 has at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:25; and (ii) The second VL of P4, which has at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:67.

[0282] 82. The protein according to any one of embodiments 29-78 and 81, (a) wherein the first heavy chain comprises: (i) The first VH of P1, which contains the sequence of SEQ ID NO:24; and (ii) The second VH of P2, which contains the sequence of SEQ ID NO:66; and (b) wherein the first light chain comprises: (i) The first VL of P3, which contains the sequence of SEQ ID NO:25; and (ii) The second VL of P4, which contains the sequence of SEQ ID NO:67.

[0283] 83. The protein according to any one of embodiments 29-31 and 33-82, wherein the first CH1 of P1 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO: 5.

[0284] 84. The protein according to any one of embodiments 29-31 and 33-83, wherein the first CH1 of P1 contains the sequence of SEQ ID NO:5.

[0285] 85. The protein according to any one of embodiments 29-31 and 33-84, wherein the second CH1 of P2 comprises a sequence having at least 75%, 80%, 85%, 90%, 95% or 99% sequence identity with the sequence of SEQ ID NO:5.

[0286] 86. The protein according to any one of embodiments 29-31 and 33-85, wherein the second CH1 of P2 comprises the sequence of SEQ ID NO:5.

[0287] 87. The protein according to any one of embodiments 29-31 and 35-86, wherein the first CL of P3 is a κ light chain constant domain.

[0288] 88. The protein according to embodiment 87, wherein the κ light chain constant domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:7.

[0289] 89. The protein according to embodiment 87 or 88, wherein the κ light chain constant domain comprises the sequence of SEQ ID NO:7.

[0290] 90. The protein according to any one of embodiments 29-31 and 38-89, wherein the second CL of P4 is a κ light chain constant domain.

[0291] 91. The protein according to embodiment 90, wherein the κ light chain constant domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:7.

[0292] 92. The protein according to embodiment 90 or 91, wherein the κ light chain constant domain comprises the sequence of SEQ ID NO:7.

[0293] 93. The protein according to any one of embodiments 29-92, wherein the first heavy chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, or SEQ ID NO:82.

[0294] 94. The protein according to any one of embodiments 29-93, wherein the first heavy chain comprises the sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81 or SEQ ID NO:82.

[0295] 95. The protein according to any one of embodiments 29-94, wherein the first light chain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:29, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:28.

[0296] 96. The protein according to any one of embodiments 29-95, wherein the first light chain comprises the sequence of SEQ ID NO:69, SEQ ID NO:72, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:29, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:28.

[0297] 97. The protein according to any one of embodiments 29-96, wherein the first heavy chain and the first light chain each comprise the following sequences: SEQ ID NO:30 and SEQ ID NO:69; SEQ ID NO:31 and SEQ ID NO:29; SEQ ID NO:75 and SEQ ID NO:68; SEQ ID NO:76 and SEQ ID NO:69; SEQ ID NO:30 and SEQ ID NO:70; SEQ ID NO:77 and SEQ ID NO:71; SEQ ID NO:78 and SEQ ID NO:72; SEQ ID NO:79 and SEQ ID NO:73; SEQ ID NO:77 and SEQ ID NO:74; SEQ ID NO:80 and SEQ ID NO:28; SEQ ID NO:81 and SEQ ID NO:68; SEQ ID NO:82 and SEQ ID NO:69; or SEQ ID NO:80 and SEQ ID NO:70.

[0298] 98. The protein according to any one of embodiments 46-97, wherein the first dimerization domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:32.

[0299] 99. The protein according to any one of embodiments 46-98, wherein the first dimerization domain comprises the sequence of SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:32.

[0300] 100. The protein according to any one of embodiments 29-99, wherein the first heavy chain comprises one or more heavy chain constant regions selected from: IgG constant region or functional fragment thereof, IgE constant region or functional fragment thereof, IgM constant region or functional fragment thereof, IgD constant region or functional fragment thereof, IgA constant region or functional fragment thereof, IgY constant region or functional fragment thereof, IgG1 constant region or functional fragment thereof, IgG2 constant region or functional fragment thereof, IgG3 constant region or functional fragment thereof, IgG4 constant region or functional fragment thereof, IgA1 constant region or functional fragment thereof, and IgA2 constant region or functional fragment thereof.

[0301] 101. The protein according to any one of embodiments 29-100, wherein the first heavy chain comprises one or more immune inert constant regions.

[0302] 102. The protein according to any one of embodiments 29-101, wherein the first heavy chain comprises one or more heavy chain constant regions selected from: wild-type human IgG1 constant region, human IgG1 constant region containing amino acid substitutions L234A, L235A and G237A, wild-type human IgG2 constant region, wild-type human IgG4 constant region and human IgG4 constant region containing amino acid substitution S228P, wherein the numbering is based on the EU index as in Kabat.

[0303] 103. The protein according to embodiment 101 or 102, wherein the heavy chain constant region comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99%, or 100% sequence identity with the sequences of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0304] 104. The protein according to any one of embodiments 29-103, further comprising a second heavy chain.

[0305] 105. The protein according to embodiment 104, wherein the second heavy chain further comprises a second dimerizing domain.

[0306] 106. The protein according to embodiment 105, wherein the second dimerization domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity with the sequence of SEQ ID NO:83, SEQ ID NO:84, or SEQ ID NO:32.

[0307] 107. The protein according to any embodiment 105 or 106, wherein the second dimerization domain comprises the sequence of SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:32.

[0308] 108. The protein according to embodiment 105, wherein the first dimerizing domain and the second dimerizing domain are dimerized.

[0309] 109. The protein according to any one of embodiments 100-104, wherein the second heavy chain comprises: (a) Second P1; (b) a third adapter, wherein the first adapter is cleavable by a protease; and (c) Second P2.

[0310] 110. The protein according to embodiment 109, further comprising a second light chain, wherein the second light chain comprises: (a) Second P3; (b) Fourth joint; and (c) Second P4.

[0311] 111. The protein according to any of the foregoing embodiments, wherein the C-terminus of the first heavy chain is connected to the N-terminus of the first dimerization domain, and the C-terminus of the first light chain is connected to the N-terminus of the second dimerization domain.

[0312] 112. A protein comprising the following structure: [P1]-[A]-[P2]-[P3]-[B]-[P4], wherein: (a) [P1] contains a first heavy chain variable domain (VH) containing a first antigen-binding domain and an optional first heavy chain constant domain 1 (CH1); (b) [A] includes a first adapter, wherein the first adapter is cleavable by a protease; (c) [P2] contains a second VH and an optional second CH1, which are second antigen-binding domains; (d) [P3] includes a first light chain variable domain (VL) and an optional first light chain constant domain (CL) comprising the first antigen-binding domain; (e) [B] includes a second connector, wherein the second connector is cleavable by a protease; (f) [P4] includes a second VL containing the second antigen-binding domain and an optional second CL; (g) [P1] and [P3] are covalently connected; (h) [P2] and [P4] are covalently connected; (i) The C terminal of [P1] is connected to the N terminal of [A]; (j) The C terminal of [A] is connected to the N terminal of [P2]; (k) The C terminal of [P3] is connected to the N terminal of [B]; (l) Connect the C terminal of [B] to the N terminal of [P4]; The first antigen-binding domain binds to PD-L1; When [A] and [B] are cleaved, the second antigen-binding domain binds to CD28.

Claims

1. A protein comprising a first polypeptide chain containing a heavy chain and a second polypeptide chain containing a light chain, The heavy chain comprises, in order from N-end to C-end, an anti-PD-L1 heavy chain variable (VH) domain, a first CH1 domain, a first joint, an anti-CD28 VH domain, and a second CH1 domain; The light chain comprises, in order 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-CD28 VL domain, and a second immunoglobulin light chain constant region.

2. The protein of claim 1, wherein the heavy chain comprises, in order from N-terminus to C-terminus, the anti-PD-L1VH domain, the first CH1 domain, the first linker, the anti-CD28 VH domain, the second CH1 domain, the hinge, the CH2 domain, and the CH3 domain.

3. The protein according to claim 1 or 2, wherein the protein further comprises a third polypeptide chain, the third polypeptide chain comprising a hinge and an Fc region.

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

32.

5. The protein according to any one of claims 1-4, wherein the first linker comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

6. The protein according to any one of claims 1-5, wherein the second linker comprises the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

7. The protein according to any one of claims 1-6, The anti-PD-L1 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:12, HCDR2 containing the amino acid sequence of SEQ ID NO:13, and HCDR3 containing the amino acid sequence of SEQ ID NO:

14. The anti-PD-L1 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:15, LCDR2 containing the amino acid sequence of SEQ ID NO:16, and LCDR3 containing the amino acid sequence of SEQ ID NO:

17. The anti-CD28 VH domain comprises HCDR1 containing the amino acid sequence of SEQ ID NO:18, HCDR2 containing the amino acid sequence of SEQ ID NO:19, and HCDR3 containing the amino acid sequence of SEQ ID NO:

20. The anti-CD28 VL domain comprises LCDR1 containing the amino acid sequence of SEQ ID NO:21, LCDR2 containing the amino acid sequence of SEQ ID NO:22, and LCDR3 containing the amino acid sequence of SEQ ID NO:

23.

8. The protein according to any one of claims 1-7, wherein the anti-PD-L1 VH domain comprises the amino acid sequence of SEQ ID NO:24, and the anti-PD-L1 VL domain comprises the amino acid sequence of SEQ ID NO:

25.

9. The protein according to any one of claims 1-8, wherein the anti-CD28 VH domain comprises the amino acid sequence of SEQ ID NO:

26.

10. The protein according to any one of claims 1-9, wherein the anti-CD28 VL domain comprises the amino acid sequence of SEQ ID NO:

27.

11. The protein according to any one of claims 1-10, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:30 or SEQ ID NO:

31.

12. The protein according to any one of claims 1-11, wherein the light chain comprises the amino acid sequence of SEQ ID NO:28 or SEQ ID NO:

29.

13. The protein according to claim 3, wherein (a) The heavy chain comprises the amino acid sequence of SEQ ID NO:30, the light chain comprises the amino acid sequence of SEQ ID NO:28, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:32; or (b) The heavy chain contains the amino acid sequence of SEQ ID NO:31, the light chain contains the amino acid sequence of SEQ ID NO:29, and the third polypeptide chain contains the amino acid sequence of SEQ ID NO:

32.

14. The protein of claim 2 or 3, wherein the heavy chain comprises a constant region of IgG, IgE, IgM, IgD, IgA or IgY.

15. The protein according to claim 2 or 3, wherein the heavy chain comprises a constant region of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

16. The protein of claim 2 or 3, wherein the heavy chain comprises an immune-inert constant region.

17. The protein according to claim 2 or 3, wherein the heavy chain comprises a wild-type human IgG1 constant region, a human IgG1 constant region comprising 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 amino acid substitution S228P, wherein the numbering is based on the EU index as in Kabat.

18. An immunoconjugate comprising a protein according to any one of claims 1-17 linked to a therapeutic agent.

19. The immunoconjugate according to claim 18, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cell inhibitory enzyme, a cell lysing enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or an apoptosis-promoting agent.

20. A pharmaceutical composition comprising a protein according to any one of claims 1-17 or an immunoconjugate according to claim 18 or 19, and a pharmaceutically acceptable carrier, diluent, or excipient.

21. A nucleic acid molecule encoding a protein according to any one of claims 1-17. (a) Heavy chain amino acid sequence; (b) Light chain amino acid sequence; or (c) Both the heavy chain amino acid sequence and the light chain amino acid sequence.

22. An expression vector comprising the nucleic acid molecule according to claim 21.

23. A recombinant host cell comprising the nucleic acid molecule according to claim 21 or the expression vector according to claim 22.

24. A method for producing a protein, the method comprising: The protein is produced by culturing a recombinant host cell containing the expression vector according to claim 22 under conditions in which the nucleic acid molecule is expressed. as well as The protein is isolated from the host cell or culture.

25. A method for enhancing an anticancer immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1-17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

26. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1-17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

27. A method for alleviating cancer symptoms in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein according to any one of claims 1-17, an immunoconjugate according to claim 18 or 19, or a pharmaceutical composition according to claim 20.

28. The method according to any one of claims 25-27, wherein the cancer is gastrointestinal stromal carcinoma (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, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, renal cell carcinoma, testicular cancer, biliary tract cancer, small bowel cancer, appendix cancer, salivary gland cancer, thyroid cancer, adrenal cancer, osteosarcoma, chondrosarcoma, or hematologic malignancy.

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