Anti-CD27 and anti-PD-L1 antibodies and bispecific constructs

Abstract

To provide an improved method for treating a subject with a condition or disease (e.g., cancer) in which stimulation of an immune response is desired.SOLUTION: Provided herein are novel anti-CD27 and anti-PD-L1 antibodies, and binding domains thereof, as well as bispecific constructs and anti-CD27 binding domains linked to an anti-PD-L1 binding domain. Also provided herein are methods of stimulating T cell activity, methods of inducing or enhancing an immune response, and methods of treating a disease or condition (e.g., cancer) by administering the bispecific constructs, antibodies, or antigen binding fragments thereof, or compositions described herein to a patient in need thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 658,899 (filed April 17, 2018) and U.S. Provisional Patent Application No. 62 / 826,091 (filed March 29, 2019). The contents of the aforementioned applications are incorporated herein by reference in their entirety. [Background technology]

[0002] The interaction between T cells and antigen-presenting cells involves various accessory molecules that facilitate the generation of an immune response. One such molecule is CD27, which binds to CD70 and belongs to the tumor necrosis factor receptor (TNF-R) superfamily (Ranheim, EA et al. (1995) Blood, 85(12):3556-65 (Non-Patent Literature 1)). CD27 typically exists as a glycosylated type I transmembrane protein, often in the form of a homodimer with a disulfide bridge linking two monomers. The disulfide bridge is located in the extracellular domain near the membrane (Camerini et al. (1991) J.Immunol., 147:3165-69 (Non-Patent Literature 2)). CD27 can also be expressed in a soluble form (see, for example, van Oers, M.H. et al. (1993) Blood 82(11):3430-6 (Non-Patent Literature 3) and Loenen, W.A. et al. (1992) Eur. J. Immunol., 22:447 (Non-Patent Literature 4)). Crosslinking of the CD27 antigen on T cells provides a co-stimulatory signal that can induce T cell proliferation and cellular immune activation in coordination with crosslinking of T cell receptors.

[0003] CD27 is expressed in mature thymocytes, most CD4+ and CD8+ peripheral blood T cells, natural killer cells, and B cells (Kobata, T. et al. (1995) Proc. Natl. Acad. Sci. USA, 92(24):11249-53 (Non-patent Literature 5)). CD27 is also highly expressed in B-cell non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia (Ranheim, EA et al. (1995) Blood, 85(12):3556-65 (Non-patent Literature 1)). In addition, increased levels of soluble CD27 protein have been observed in the serum or disease-active sites of parasitic infections, cytomegalovirus (CMV) infections, sarcoidosis, multiple sclerosis, and B-cell chronic lymphocytic leukemia (Loenen, WA et al. (1992) Eur. J. Immunol, 22:447 (Non-patent Literature 4)).

[0004] Programmed death ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein that is thought to play a major role in suppressing the immune system during certain events such as pregnancy, tissue allogeneic transplantation, autoimmune diseases, and other medical conditions (such as hepatitis). Normally, the immune system responds to foreign antigens associated with exogenous or endogenous danger signals, which trigger the proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells. PD-L1 binding to PD-1 transmits an inhibitory signal that reduces the proliferation of these T cells and can also induce apoptosis, which is further mediated by lower regulation of the Bcl-2 gene. PD-L1 is present in large quantities in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9 (Non-Patent Literature 6)). The interaction between PD-1 and PD-L1 results in a reduction of tumor-infiltrating lymphocytes, decreased T-cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J.Mol.Med.81:281-7 (Non-Patent Literature 7), Blank et al. (2005) Cancer Immunol.Immunother.54:307-314 (Non-Patent Literature 8), Konishi et al. (2004) Clin.Cancer Res.10:5094-100 (Non-Patent Literature 9)). By inhibiting the local interaction between PD-1 and PD-L1, immunosuppression can be reversed, and if the interaction between PD-1 and PD-L2 is similarly blocked, this effect is additive (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7 (Non-patent Literature 10), Brown et al. (2003) J. Immunol. 170:1257-66 (Non-patent Literature 11)).

[0005] Despite advances in multidisciplinary therapy, there is a need in the art for new and improved therapeutic agents for treating conditions or diseases (e.g., those in which stimulation of the immune response is desired). Therefore, an object of the present invention is to provide an improved method for treating subjects having such conditions or diseases (e.g., cancer). [Prior art documents]

Non-Patent Literature

[0006]

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Summary of the Invention

[0007] Novel anti-CD27 and anti-PD-L1 antibodies, as well as their binding domains, and bispecific and multispecific constructs comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain are provided herein. Methods for stimulating T cell activity, inducing or enhancing an immune response, and doing so by administering the bispecific or multispecific constructs, antibodies, or their antigen-binding fragments or compositions described herein to patients who require treatment for a disease or condition (e.g., cancer).

[0008] An exemplary anti-CD27 antibody is antibody 3C2 as described herein. In one embodiment, the anti-CD27 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 3C2. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 3C2 having the sequence described in SEQ ID NO: 17, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 3C2 having the sequence described in SEQ ID NO: 18. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains having the sequences described in SEQ ID NOs: 1, 2 and 3, respectively, or their conserved sequence modifications, and the light chain CDR1, CDR2 and CDR3 domains having the sequences described in SEQ ID NOs: 4, 5 and 6, respectively, or their conserved sequence modifications. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 17. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 18. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0009] Another exemplary anti-CD27 antibody is antibody 2B3 as described herein. In one embodiment, the anti-CD27 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 2B3. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 2B3 having the sequence described in SEQ ID NO: 19, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 3C2 having the sequence described in SEQ ID NO: 20. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 7, 8 and 9, respectively, and the light chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 10, 11 and 12, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 19. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 20. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0010] An exemplary anti-PD-L1 antibody is antibody 7H7 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 7H7. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 7H7 having the sequence described in SEQ ID NO: 77, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 7H7 having the sequence described in SEQ ID NO: 78. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 29, 30 and 31, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 32, 33 and 34, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 77. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 77. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively.

[0011] Another exemplary anti-PD-L1 antibody is antibody 1B3 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 1B3. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 1B3 having the sequence described in SEQ ID NO: 79, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 1B3 having the sequence described in SEQ ID NO: 80. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 35, 36 and 37, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 38, 39 and 40, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 79. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 80. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively.

[0012] Another exemplary anti-PD-L1 antibody is antibody 3B6 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 3B6. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 3B6 having the sequence described in SEQ ID NO: 81, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 3B6 having the sequence described in SEQ ID NO: 82. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 41, 42 and 43, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 44, 45 and 46, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 81. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 82. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively.

[0013] Another exemplary anti-PD-L1 antibody is antibody 8B1 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 8B1. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 8B1 having the sequence described in SEQ ID NO: 83, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 8B1 having the sequence described in SEQ ID NO: 84. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 47, 48 and 49, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 50, 51 and 52, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 83. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 84. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.

[0014] Another exemplary anti-PD-L1 antibody is antibody 4A3 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 4A3. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 4A3 having the sequence described in SEQ ID NO: 85, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 4A3 having the sequence described in SEQ ID NO: 86. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 56, 57 and 58, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 85. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 86. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively.

[0015] Another exemplary anti-PD-L1 antibody is antibody 9H9 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 9H9. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 9H9 having the sequence described in SEQ ID NO: 87, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 9H9 having the sequence described in SEQ ID NO: 88. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 59, 60 and 61, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 62, 63 and 64, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 87. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 88. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively.

[0016] In one embodiment, the CDR1, 2, and / or 3 regions of the anti-C27 or anti-PD-L1 binding domain described herein may contain the exact same amino acid sequence as the antibodies 3C2, 2B3, 7H7, 1B3, 3B6, 8B1, 4A3, and 9H9 disclosed herein. In another embodiment, the antibody contains derivatives from the exact same CDR sequences as 3C2, 2B3, 7H7, 1B3, 3B6, 8B1, 4A3, and 9H9, but still retains the ability to effectively bind to either CD27 or PD-L1. Such sequence modifications may include the addition, deletion, or substitution of one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acids, such as conservative sequence modifications.

[0017] In another embodiment, the anti-C27 or anti-PD-L1 binding domain described herein may consist of one or more CDRs that are 90%, 95%, 98%, or 99.5% identical to one or more CDRs of the antibodies 3C2, 2B3, 7H7, 1B3, 3B6, 8B1, 4A3, and 9H9. CDRs having an identity in the intermediate range of the values ​​listed above, for example, 90-95%, 95-98%, or 98-100% identical to one or more of the sequences above, are also intended to be included by the present invention.

[0018] The antibody sequence may also be the consensus sequence of several antibodies. For example, in one embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region CDR1 containing an amino acid sequence selected from the consensus sequence: (T,S)(S,Y,H)WMS (SEQ ID NO: 167). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region CDR2 containing SEQ ID NO: 168. In yet another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region CDR3 containing SEQ ID NO: 169. In yet another embodiment, the anti-PD-L1 binding domain includes a light chain variable region CDR1 containing SEQ ID NO: 170. In yet another embodiment, the anti-PD-L1 binding domain includes a light chain variable region CDR2 containing SEQ ID NO: 171. In yet another embodiment, the anti-PD-L1 binding domain includes a light chain variable region CDR3 containing SEQ ID NO: 172.

[0019] Sequences substantially identical to the anti-C27 and / or anti-PD-L1 binding domains described herein (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences) are also included by the present invention. In one embodiment, the anti-CD27 binding domain includes a heavy chain variable region containing sequence number 17, sequence number 19, or at least 90% identical to them (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences). In another embodiment, the anti-CD27 binding domain includes a light chain variable region containing sequence number 18, sequence number 20, or a sequence that is at least 90% identical to them (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences). In another embodiment, the anti-CD27 binding domain includes a heavy chain variable region containing sequence number 17 or a sequence that is at least 90% identical to it (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences), and a light chain variable region containing sequence number 18 or a sequence that is at least 90% identical to it (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences). In another embodiment, the anti-CD27 binding domain includes a heavy chain variable region containing the sequence of sequence number 19 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of sequence number 20 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0020] In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, or a sequence that is at least 90% identical to them (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences). In another embodiment, the anti-PD-L1 binding domain includes a light chain variable region containing sequence SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or a sequence that is at least 90% identical to them (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 77 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 78 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing the sequence of SEQ ID NO: 79 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 80 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 81 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 82 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 83 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 84 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 85 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 86 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 87 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 88 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0021] Anti-CD27 and / or anti-PD-L1 antibodies or their binding domains that compete for binding with any of the antibodies or their binding domains described herein, or that bind to the same epitope as any of the antibodies or their binding domains described herein, are also suitable for use and are included by the present invention. For example, in one embodiment, an anti-CD27 antibody or its binding domain competes with antibody 3C2 and / or antibody 2B3 for binding to CD27, as described herein. For example, as described in Example 28, the antibody of the present invention (e.g., antibody 2B3) binds to one or more residues in amino acids 80-95 of the ECD of human CD27 (SEQ ID NO: 183), for example, one or more residues in amino acids 85-89 of the ECD of human CD27 (SEQ ID NO: 183), for example, one or more residues in amino acids 85, 87, 88 and / or 89.

[0022] In another embodiment, the antibody binds to the wild-type ECD of human CD27 but does not bind to mutant ECDs of human CD27 (SEQ ID NO: 183) that have amino acid substitutions at one or more positions within amino acid residues 85-89 (e.g., A85S, R87A, N88A, and / or G89A). For example, an anti-CD27 antibody or its antigen-binding fragment binds to the wild-type ECD of human CD27 (SEQ ID NO: 183) but does not bind to mutant human CD27 wild-type ECDs having the following amino acid substitutions: A85S, R87A, N88A, and G89A.

[0023] In another embodiment, the anti-CD27 antibody or binding domain binds to the same epitope on CD27 as antibody 3C2 and / or antibody 2B3, as described herein. In another embodiment, the antibody or anti-PD-L1 binding domain competes with antibodies 7H7, 1B3, 3B6, 8B1, 4A3 and / or 9H9 for binding to PD-L1, as described herein. In another embodiment, the anti-PD-L1 antibody or binding domain binds to the same epitope on PD-L1 as antibodies 7H7, 1B3, 3B6, 8B1, 4A3 and / or 9H9, as described herein.

[0024] In one embodiment, a bispecificity construct (or multispecificity construct) comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain, (i) The anti-CD27 binding domain is a. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3 respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6 respectively, or their conservative sequence modifications, b. Including the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, or their conserved sequence modifications, (ii) The anti-PD-L1 binding domain is a. Heavy chain variable region CDR1 containing an amino acid sequence selected from the consensus sequence (T,S)(S,Y,H)WMS (SEQ ID NO: 167), b. Heavy chain variable region CDR2 including sequence number 168, c. Heavy chain variable region CDR3 including sequence number 169, d. Light chain variable region CDR1 including sequence number 170, e. Light chain variable region CDR2 including sequence number 171, A bispecific construct is provided that includes a light chain variable region CDR3 containing sequence number 172.

[0025] In another embodiment, a bispecific construct comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain, (i) The anti-CD27 binding domain is a. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3 respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6 respectively, or their conservative sequence modifications, b. Including the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, or their conserved sequence modifications, (ii) The anti-PD-L1 binding domain is a. A heavy chain variable region containing sequence number 77 or at least 95% identical thereto, and a light chain variable region containing sequence number 78 or at least 95% identical thereto, b. A heavy chain variable region containing sequence number 79 or at least 95% identical thereto, and a light chain variable region containing sequence number 80 or at least 95% identical thereto, c. A heavy chain variable region containing sequence number 81 or a sequence that is at least 95% identical thereto, and a light chain variable region containing sequence number 82 or a sequence that is at least 95% identical thereto, d. A heavy chain variable region containing sequence number 83 or a sequence that is at least 95% identical thereto, and a light chain variable region containing sequence number 84 or a sequence that is at least 95% identical thereto, e. A heavy chain variable region containing sequence number 85 or a sequence that is at least 95% identical thereto, and a light chain variable region containing sequence number 86 or a sequence that is at least 95% identical thereto, f. A bispecific construct comprising a heavy chain variable region containing sequence number 87 or at least 95% identical thereto, and a light chain variable region containing sequence number 88 or at least 95% identical thereto.

[0026] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 29, 30, and 31, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 32, 33, and 34, respectively.

[0027] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region comprising SEQ ID NO: 77 and a light chain variable region comprising SEQ ID NO: 78.

[0028] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 35, 36, and 37, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 38, 39, and 40, respectively.

[0029] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80.

[0030] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 41, 42, and 43, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 44, 45, and 46, respectively.

[0031] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82.

[0032] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 50, 51, and 52, or their conserved sequence modifications.

[0033] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region comprising SEQ ID NO: 83 and a light chain variable region comprising SEQ ID NO: 84.

[0034] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 56, 57, and 58, or their conserved sequence modifications.

[0035] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86.

[0036] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications, and an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 62, 63, and 64, or their conserved sequence modifications.

[0037] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0038] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, or their conserved sequence modifications.

[0039] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 77 and a light chain variable region containing SEQ ID NO: 78.

[0040] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, or their conserved sequence modifications.

[0041] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80.

[0042] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, or their conserved sequence modifications.

[0043] In another embodiment, the bispecific construct includes (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82.

[0044] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications.

[0045] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84.

[0046] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, or their conserved sequence modifications.

[0047] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86.

[0048] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications.

[0049] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0050] In one embodiment, the anti-PD-L1 binding domain and the anti-CD27 binding domain are genetically fused. The bispecific construct may be a fusion protein that can be produced, for example, by genetically engineering using standard recombinant DNA techniques to operably link nucleic acids encoding the anti-CD27 and anti-PD-L1 binding domains. In another embodiment, the anti-PD-L1 binding domain and the anti-CD27 binding domain are chemically coupled.

[0051] For example, a bispecific construct may be a chemical conjugate that can be prepared by chemically conjugating anti-CD27 and anti-PD-L1 binding domains. In one embodiment, the anti-PD-L1 binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-CD27 binding domain is ligated to the C-terminus of the heavy chain of the anti-PD-L1 binding domain. In yet another embodiment, the anti-CD27 binding domain is scFv.

[0052] In another embodiment, the anti-CD27 binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-PD-L1 binding domain is ligated to the C-terminus of the heavy chain of the anti-CD27 binding domain. In another embodiment, the anti-PD-L1 binding domain is scFv.

[0053] In certain embodiments, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv. (i) Anti-CD27 scFv is, a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 4, 5, and 6, respectively, or b. comprising heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively. (ii) Anti-PD-L1 antibodies are a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 29, 30, and 31, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 32, 33, and 34, respectively. b. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 35, 36, and 37, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 38, 39, and 40, respectively. c. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 41, 42, and 43, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 44, 45, and 46, respectively. d. Heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 50, 51, and 52, respectively. e. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 53, 54, and 55, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 56, 57, and 58, respectively, or f. Heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively. g. Contains the human IgG1 constant domain.

[0054] In another specific embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) Anti-CD27 antibody, a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 4, 5, and 6, respectively, or b. Heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 10, 11, and 12, respectively, c. Includes the human IgG1 constant domain, (ii) Anti-PD-L1 scFv is, a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 29, 30, and 31, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 32, 33, and 34, respectively. b. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 35, 36, and 37, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 38, 39, and 40, respectively. c. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 41, 42, and 43, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 44, 45, and 46, respectively. d. Heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 50, 51, and 52, respectively. e. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 53, 54, and 55, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 56, 57, and 58, respectively, or f. Includes heavy chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 59, 60, and 61, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 62, 63, and 64, respectively.

[0055] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv is, a. Heavy chain variable region including SEQ ID NO: 17 and light chain variable region including SEQ ID NO: 18, or b. Includes a heavy chain variable region containing Sequence ID 19 and a light chain variable region containing Sequence ID 20, (ii) Anti-PD-L1 antibodies are a. Heavy chain variable region including Sequence ID 77 and light chain variable region including Sequence ID 78, b. Heavy chain variable region containing Sequence ID 79 and light chain variable region containing Sequence ID 80, c. Heavy chain variable region including SEQ ID NO. 81 and light chain variable region including SEQ ID NO. 82, d. Heavy chain variable region including SEQ ID NO: 83 and light chain variable region including SEQ ID NO: 84, e. Heavy chain variable region containing Sequence ID 85 and light chain variable region containing Sequence ID 86, or f. A heavy chain variable region including sequence number 87 and a light chain variable region including sequence number 88, g. Contains the human IgG1 constant domain.

[0056] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) Anti-CD27 antibody, a. Heavy chain variable region including SEQ ID NO: 17 and light chain variable region including SEQ ID NO: 18, or b. A heavy chain variable region containing Sequence ID 19 and a light chain variable region containing Sequence ID 20, c. Includes the human IgG1 constant domain, (ii) Anti-PD-L1 scFv is, a. Heavy chain variable region including Sequence ID 77 and light chain variable region including Sequence ID 78, b. Heavy chain variable region containing Sequence ID 79 and light chain variable region containing Sequence ID 80, c. Heavy chain variable region including SEQ ID NO. 81 and light chain variable region including SEQ ID NO. 82, d. Heavy chain variable region including SEQ ID NO: 83 and light chain variable region including SEQ ID NO: 84, e. Heavy chain variable region containing Sequence ID 85 and light chain variable region containing Sequence ID 86, or f. Includes a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0057] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 10, 11, and 12, respectively. (ii) The PD-L1 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively, and the human IgG1 constant domain.

[0058] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively, and the human IgG1 constant domain. (ii) Anti-PD-L1 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively.

[0059] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20 (ii) The anti-PD-L1 antibody comprises a heavy chain variable region containing SEQ ID NO: 83, a light chain variable region containing SEQ ID NO: 84, and a human IgG1 constant domain.

[0060] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises a heavy chain variable region containing SEQ ID NO: 19, a light chain variable region containing SEQ ID NO: 20, and a human IgG1 constant domain. (ii) The anti-PD-L1 scFv comprises a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84.

[0061] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 10, 11, and 12, respectively. (ii) The anti-PD-L1 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively, and the human IgG1 constant domain.

[0062] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively, and the human IgG1 constant domain. (ii) Anti-PD-L1 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively.

[0063] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20 (ii) The anti-PD-L1 antibody comprises a heavy chain variable region containing SEQ ID NO: 87, a light chain variable region containing SEQ ID NO: 88, and a human IgG1 constant domain.

[0064] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises a heavy chain variable region containing SEQ ID NO: 19, a light chain variable region containing SEQ ID NO: 20, and a human IgG1 constant domain. (ii) The anti-PD-L1 scFv comprises a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0065] In another embodiment, the bispecific construct has one or more of the following functional features: inducing NFκB activation, increasing T cell proliferation, inducing a CD8 T cell response, and / or increasing IL-2 production. In another embodiment, the bispecific construct increases IL-2 production by at least about 1.5 times (e.g., at least 2 times, 2.5 times, 3 times, 3.5 times, or 4 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone. In another embodiment, the bispecific construct induces a CD8 T cell response at least about 2 times greater (e.g., at least 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8.0 times, 8.5 times, or 9 times) than anti-CD27 monoclonal antibody alone. In another embodiment, the bispecific construct increases survival by at least approximately 1.5 times (e.g., at least 1.5 times, 2.0 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone or in combination. In another embodiment, the bispecific construct reduces tumor weight by at least approximately 1.5 times (e.g., at least 1.5 times, 2.0 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone or in combination. In another embodiment, the bispecific construct increases T cell production by at least about 1.5 times (e.g., at least 1.5 times, 2.0 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8.0 times, 8.5 times, or 9 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone or in combination.

[0066] In certain embodiments, the bispecific constructs described herein exhibit synergistic effects (e.g., in enhancing the immune response in vivo) compared to the use of a combination of anti-CD27 binding domains and anti-PD-L1 binding domains (i.e., co-administration of unbound antibodies).

[0067] In another embodiment, a novel anti-CD27 antibody or its antigen-binding moiety is provided, comprising any of the anti-CD27 binding domains described herein. In one embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises a heavy chain variable region comprising a sequence of SEQ ID NO: 17 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence), and a light chain variable region comprising a sequence of SEQ ID NO: 18 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0068] In another embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively. In another embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NOs: 19 or a sequence at least 95% identical thereto, and a light chain variable region containing SEQ ID NOs: 20 or a sequence at least 95% identical thereto.

[0069] In another embodiment, the anti-CD27 antibody or its antigen-binding fragment has the following functional characteristics: it induces or enhances a T cell-mediated immune response; it blocks (e.g., partially or completely) the binding of sCD70 to CD27; it induces NFκB activation; it increases T cell proliferation; and it has an equilibrium dissociation constant Kd of 10. -9 M is less than or equal to 10, or the equilibrium association constant Ka is 10 +9 M -1The above describes a functional characteristic which is one or more of the following: binding to human CD27; inducing specific complement-mediated cytotoxicity (CDC) in CD27-expressing cells; inducing antibody-dependent cell-mediated cytotoxicity (ADCC) specific lysis in CD27-expressing cells; inducing or enhancing an antigen-specific immune response in vivo in combination with a vaccine or endogenous antigen; inducing or enhancing an antigen-specific TH1 immune response in vivo in combination with a vaccine or endogenous antigen; inducing or enhancing antigen-specific T cell proliferation or activation in vivo in combination with a vaccine or endogenous antigen; and / or inducing or enhancing T cell activity when combined with simultaneous, separate, or sequential TCR activation.

[0070] In another embodiment, a bispecific construct is provided, comprising any one of the anti-CD27 antibodies described herein ligated to an anti-PD-L1 binding domain. In one embodiment, the anti-PD-L1 binding domain is a. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively. b. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, respectively. c. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, respectively. d. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively. e. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, respectively, and f. Selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding fragment, comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively.

[0071] In another embodiment, the anti-PD-L1 binding domain is selected from the group consisting of (a) a heavy chain variable region including SEQ ID NO: 77 and a light chain variable region including SEQ ID NO: 78, (b) a heavy chain variable region including SEQ ID NO: 79 and a light chain variable region including SEQ ID NO: 80, (c) a heavy chain variable region including SEQ ID NO: 81 and a light chain variable region including SEQ ID NO: 82, (d) a heavy chain variable region including SEQ ID NO: 83 and a light chain variable region including SEQ ID NO: 84, (e) a heavy chain variable region including SEQ ID NO: 85 and a light chain variable region including SEQ ID NO: 86, and (f) a heavy chain variable region including SEQ ID NO: 87 and a light chain variable region including SEQ ID NO: 88. In a particular embodiment, the anti-PD-L1 binding domain is scFv.

[0072] In certain embodiments, the bispecific constructs described herein exhibit synergistic effects (e.g., in enhancing the immune response in vivo) compared to the use of a combination of anti-CD27 binding domains and anti-PD-L1 binding domains (i.e., co-administration of unbound antibodies).

[0073] In another embodiment, a novel anti-PD-L1 antibody or its antigen-binding moiety is provided, comprising any of the anti-PD-L1 binding domains described herein. In one embodiment, the anti-PD-L1 antibody or its antigen-binding fragment comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing the sequence of SEQ ID NO: 77 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 78 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 38, 39, and 40, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 79 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 80 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 41, 42, and 43, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 44, 45, and 46, respectively.In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 81 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 82 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 50, 51, and 52, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 83 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 84 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 53, 54, and 55, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 56, 57, and 58, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 85 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 86 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment comprises a heavy chain variable region containing a sequence of SEQ ID NO. 87 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO. 88 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0074] In another embodiment, an anti-PD-L1 antibody or its antigen-binding fragment has one or more of the following functional features: (a) blocking the binding of PD1 to PD-L1 (e.g., partially or completely), (b) inducing NFAT pathway activation, and / or (c) inducing a mixed lymphocyte response.

[0075] In another embodiment, a bispecific construct is provided, comprising one of the anti-PD-L1 antibodies described herein or their antigen-binding fragments, ligated to an anti-CD27 binding domain. In one embodiment, the anti-CD27 binding domain comprises an anti-CD27 antibody comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the anti-CD27 binding domain comprises an antibody comprising a heavy chain variable region containing the sequence of SEQ ID NO: 17 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 18 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-CD27 binding domain comprises an anti-CD27 antibody comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively. In another embodiment, the anti-C27 binding domain comprises a CD27 antibody comprising a heavy chain variable region containing the sequence of SEQ ID NO: 19 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 20 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In one embodiment, the anti-CD27 binding domain further comprises a human IgG1 constant domain.

[0076] In certain embodiments, the bispecific constructs described herein exhibit synergistic effects (e.g., in enhancing the immune response in vivo) compared to the use of a combination of anti-CD27 binding domains and anti-PD-L1 binding domains (i.e., co-administration of unbound antibodies).

[0077] In another embodiment, compositions comprising any of the bispecific constructs (multispecific constructs), antibodies, or antigen-binding fragments thereof described herein, and a pharmaceutically acceptable carrier are provided herein. Kits comprising any of the bispecific constructs (multispecific constructs), antibodies, or antigen-binding fragments thereof described herein, and instructions for use are also provided herein.

[0078] In further embodiments, isolated nucleic acid molecules encoding binding domains, antibodies, or their antigen-binding moieties, as well as the bispecific or multispecific constructs described herein, and expression vectors containing such nucleic acids and host cells containing such expression vectors are also provided. In another embodiment, nucleic acid molecules encoding any of the binding domains, antibodies, or their antigen-binding moieties, or the bispecific constructs described herein are provided. In another embodiment, the nucleic acid molecule is in the form of an expression vector. In another embodiment, the nucleic acid molecule is in the form of an expression vector or a bispecific construct that, when administered in vivo to a subject, expresses a binding domain, an antibody, or its antigen-binding moiety.

[0079] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody variable region, the antibody variable region comprising an amino acid sequence shown in SEQ ID NOs: 17, 18, 19, 20, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or an amino acid sequence that is at least 90% identical thereto (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the aforementioned sequences). In another embodiment, the nucleic acid molecule includes a nucleotide sequence described in SEQ ID NOs: 25, 26, 27, 28, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or a nucleotide sequence that is at least 90% identical thereto (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the aforementioned sequences).

[0080] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain and light chain variable regions of an antibody, the heavy chain and light chain variable regions comprising the amino acid sequence shown in SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20, SEQ ID NOs. 77 and 78, SEQ ID NOs. 79 and 80, SEQ ID NOs. 81 and 82, SEQ ID NOs. 83 and 84, SEQ ID NOs. 85 and 86, or SEQ ID NOs. 87 and 88, respectively, or an amino acid sequence that is at least 90% identical thereto (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0081] In another embodiment, a method is provided for stimulating T cell activity, comprising contacting T cells with an antibody, or one of its antigen-binding fragments, bispecific constructs, multispecific constructs, or compositions described herein. Stimulating T cell activity may include, for example, stimulating IFN-gamma production.

[0082] In yet another embodiment, a method for inducing or enhancing an immune response (e.g., to an antigen) in a subject comprises administering to the subject an antibody, or one of its antigen-binding fragments, bispecific constructs, multispecific constructs, or compositions described herein, in an amount effective to induce or enhance an immune response (e.g., to an antigen) in the subject.

[0083] In a further embodiment, a method is provided for treating a condition or disease in a subject, the method comprising administering to the subject an antibody, or one thereof, an antigen-binding fragment, a bispecific construct, a multispecific construct, or a composition described herein, in an amount effective for treating the condition or disease.

[0084] In another embodiment, a method is provided for treating a condition or disease in a subject, the method comprising administering to the subject one of the anti-CD27 antibodies or their antigen-binding fragments described herein in combination with one of the anti-PD-L1 antibodies or their antigen-binding fragments described herein. For example, in one embodiment, (i) The anti-CD27 antibody or its antigen-binding fragment is selected from the group consisting of (a) an anti-CD27 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6, respectively, and (b) an anti-CD27 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively. (ii) The anti-PD-L1 antibody or its antigen-binding fragment comprises (a) the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively; (b) the anti-PD-L1 antibody or its antigen-binding fragment comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, respectively; (c) the anti-PD-L1 antibody or its antigen-binding fragment comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, respectively. A fragment is selected from the group consisting of (d) an anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively; (e) an anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, respectively; and (f) an anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively.

[0085] In another embodiment, (i) The anti-CD27 antibody or its antigen-binding fragment is selected from the group consisting of (a) an anti-CD27 antibody or its antigen-binding fragment comprising a heavy chain variable region including SEQ ID NO: 17 and a light chain variable region including SEQ ID NO: 18, and (b) an anti-CD27 antibody or its antigen-binding fragment comprising a light chain variable region including SEQ ID NO: 19 and SEQ ID NO: 20 or a sequence, (ii) The anti-PD-L1 antibody or its antigen-binding fragment is selected from the group consisting of: (a) an anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 77 and a light chain variable region comprising SEQ ID NO: 78; (b) an anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 79 and a light chain variable region comprising SEQ ID NO: 80; (c) an anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 81 and a light chain variable region comprising SEQ ID NO: 82; (d) an anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 83 and a light chain variable region comprising SEQ ID NO: 84; (e) an anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 85 and a light chain variable region comprising SEQ ID NO: 86; and (f) an anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region comprising SEQ ID NO: 87 and a light chain variable region comprising SEQ ID NO: 88.

[0086] In one embodiment, the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are administered separately. In another embodiment, the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are administered sequentially. For example, the anti-CD27 antibody or its antigen-binding fragment is administered first, followed (for example, immediately afterward) by the anti-PD-L1 antibody or its antigen-binding fragment, or vice versa. In yet another embodiment, the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are administered together. In yet another embodiment, the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are administered simultaneously. In yet another embodiment, the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are administered simultaneously in a single formulation. Alternatively, the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are formulated for separate administration and administered simultaneously or sequentially. Such simultaneous or sequential administration preferably results in both antibodies being present at the same time in the patient being treated.

[0087] In certain embodiments, administration of any of the anti-CD27 antibodies or their antigen-binding fragments described herein in combination with any of the anti-PD-L1 antibodies or their antigen-binding fragments described herein results in a synergistic effect (e.g., in enhancing the immune response in vivo) compared to the use of either antibody alone.

[0088] The subjects may be, for example, individuals suffering from a condition or disease in which stimulation of the immune response is desired. In one embodiment, the condition or disease in which stimulation of the immune response is desired is cancer. A method for inducing or enhancing an immune response (e.g., against an antigen) in a subject may further include administering an antigen to the subject. Antigens that are preferably administered co-administered with antibodies or their antigen-binding fragments, bispecific constructs, multispecific constructs, or compositions described herein are tumor antigens. [Invention 1001] A bispecific construct comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain, (i) The anti-CD27 binding domain is a. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3 respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6 respectively, or their conservative sequence modifications, b. Including the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, or their conserved sequence modifications, (ii) The anti-PD-L1 binding domain is a. A heavy chain variable region CDR1 containing an amino acid sequence selected from the consensus sequence (T,S)(S,Y,H)WMS (SEQ ID NO: 167) or its conserved sequence modifications, b. Heavy chain variable region CDR2 containing sequence number 168 or its conservative sequence modification, c. Heavy chain variable region CDR3 containing sequence number 169 or its conservative sequence modification, d. Light chain variable region CDR1 containing sequence number 170 or its conservative sequence modification, e. Light chain variable region CDR2 containing sequence number 171 or its conservative sequence modification, f. A bispecific construct comprising the light chain variable region CDR3 containing sequence number 172 or a conservative sequence modification thereof. [Invention 1002] A bispecific construct comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain, (i) The anti-CD27 binding domain is a. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3 respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6 respectively, or their conservative sequence modifications, b. Including the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, or their conserved sequence modifications, (ii) The anti-PD-L1 binding domain is a. A heavy chain variable region containing sequence number 77 or at least 95% identical thereto, and a light chain variable region containing sequence number 78 or at least 95% identical thereto, b. A heavy chain variable region containing sequence number 79 or at least 95% identical thereto, and a light chain variable region containing sequence number 80 or at least 95% identical thereto, c. A heavy chain variable region containing sequence number 81 or a sequence that is at least 95% identical thereto, and a light chain variable region containing sequence number 82 or a sequence that is at least 95% identical thereto, d. A heavy chain variable region containing sequence number 83 or a sequence that is at least 95% identical thereto, and a light chain variable region containing sequence number 84 or a sequence that is at least 95% identical thereto, e. A heavy chain variable region containing sequence number 85 or a sequence that is at least 95% identical thereto, and a light chain variable region containing sequence number 86 or a sequence that is at least 95% identical thereto, f. A bispecific construct comprising a heavy chain variable region containing sequence number 87 or at least 95% identical thereto, and a light chain variable region containing sequence number 88 or at least 95% identical thereto. [Invention 1003] A bispecific construct of the present invention 1001 or 1002, wherein the anti-CD27 binding domain comprises a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18. [Invention 1004] A bispecific construct of the present invention 1001 or 1002, wherein the anti-CD27 binding domain comprises a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20. [Invention 1005] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, or their conserved sequence modifications. [Invention 1006] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 77 and a light chain variable region containing SEQ ID NO: 78. [Invention 1007] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, or their conserved sequence modifications. [Invention 1008] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80. [Invention 1009] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, or their conserved sequence modifications. [Invention 1010] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82. [Invention 1011] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications. [Invention 1012] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84. [Invention 1013] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, or their conserved sequence modifications. [Invention 1014] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86. [Invention 1015] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications. [Invention 1016] A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88. [Invention 1017] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, or their conserved sequence modifications. [Invention 1018] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 77 and a light chain variable region containing SEQ ID NO: 78. [Invention 1019] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, or their conserved sequence modifications. [Invention 1020] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80. [Invention 1021] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, or their conserved sequence modifications. [Invention 1022] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82. [Invention 1023] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications. [Invention 1024] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84. [Invention 1025] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, or their conserved sequence modifications. [Invention 1026] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86. [Invention 1027] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications. [Invention 1028] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88. [Invention 1029] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, or their conserved sequence modifications. [Invention 1030] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 77 and a light chain variable region containing SEQ ID NO: 78. [Invention 1031] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, or their conserved sequence modifications. [Invention 1032] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80. [Invention 1033] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, or their conserved sequence modifications. [Invention 1034] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82. [Invention 1035] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications. [Invention 1036] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84. [Invention 1037] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, or their conserved sequence modifications. [Invention 1038] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86. [Invention 1039] a. The anti-CD27 binding domain comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, or their conserved sequence modifications. b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications. [Invention 1040] a. The anti-CD27 binding domain includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, b. A bispecific construct of the present invention 1001 or 1002, wherein the anti-PD-L1 binding domain comprises a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88. [Invention 1041] (a) a bispecific construct of any of the invention 1001 to 1040, wherein the anti-PD-L1 binding domain further comprises a human IgG1 constant domain, or (b) the anti-CD27 binding domain further comprises a human IgG1 constant domain. [Invention 1042] (a) The anti-CD27 binding domain is ligated to the C-terminus of the heavy chain of the anti-PD-L1 binding domain, or (b) The anti-PD-L1 binding domain is ligated to the C-terminus of the heavy chain of the anti-CD27 binding domain, a bispecific construct according to any of the invention 1001 to 1041. [Invention 1043] A bispecific construct according to any of the invention 1001 to 1042, wherein (b) the anti-CD27 binding domain is scFv, or (b) the anti-PD-L1 binding domain is scFv. [Invention 1044] A bispecific construct according to any one of the invention 1001 to 1043, wherein the anti-PD-L1 binding domain and the anti-CD27 binding domain are genetically fused. [Invention 1045] A bispecific construct according to any of the present invention 1001 to 1044, wherein the anti-PD-L1 binding domain and the anti-CD27 binding domain are chemically conjugated. [Invention 1046] A bispecific construct comprising an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) The anti-CD27 scFv is, a. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3 respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6 respectively, or their conservative sequence modifications, b. Including the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, or their conserved sequence modifications, (ii) The anti-PD-L1 antibody is a. Heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, respectively, or their conserved sequence modifications, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively, or their conserved sequence modifications. b. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 35, 36, and 37, respectively, or their conserved sequence modifications, and light chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 38, 39, and 40, respectively, c. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, respectively, or their conservative sequence modifications. d. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively, or their conserved sequence modifications. e. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, respectively, or their conservative sequence modifications, f. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively, or their conserved sequence modifications. g. A bispecific construct containing the human IgG1 constant domain. [Invention 1047] A bispecific construct comprising an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) The anti-CD27 scFv is, a. Heavy chain variable region including SEQ ID NO: 17 and light chain variable region including SEQ ID NO: 18, or b. Includes a heavy chain variable region containing Sequence ID 19 and a light chain variable region containing Sequence ID 20, (ii) The anti-PD-L1 antibody is a. Heavy chain variable region including Sequence ID 77 and light chain variable region including Sequence ID 78, b. Heavy chain variable region containing Sequence ID 79 and light chain variable region containing Sequence ID 80, c. Heavy chain variable region including SEQ ID NO. 81 and light chain variable region including SEQ ID NO. 82, d. Heavy chain variable region including SEQ ID NO: 83 and light chain variable region including SEQ ID NO: 84, e. Heavy chain variable region containing Sequence ID 85 and light chain variable region containing Sequence ID 86, or f. A heavy chain variable region including sequence number 87 and a light chain variable region including sequence number 88, g. A bispecific construct containing the human IgG1 constant domain. [Invention 1048] A bispecific construct comprising an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) The anti-CD27 scFv comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conservative sequence modifications, (ii) A bispecific construct comprising the anti-PD-L1 antibody, each comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications, and the human IgG1 constant domain. [Invention 1049] A bispecific construct comprising an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) The anti-CD27 scFv includes a heavy chain variable region containing sequence number 19 and a light chain variable region containing sequence number 20, (ii) A bispecific construct comprising the anti-PD-L1 antibody, a heavy chain variable region containing SEQ ID NO: 83, a light chain variable region containing SEQ ID NO: 84, and a human IgG1 constant domain. [Invention 1050] A bispecific construct comprising an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) The anti-CD27 scFv comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conservative sequence modifications, (ii) A bispecific construct comprising the anti-PD-L1 antibody, each comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications, and the human IgG1 constant domain. [Invention 1051] A bispecific construct comprising an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) The anti-CD27 scFv includes a heavy chain variable region containing sequence number 19 and a light chain variable region containing sequence number 20, (ii) A bispecific construct comprising the anti-PD-L1 antibody, a heavy chain variable region containing SEQ ID NO: 87, a light chain variable region containing SEQ ID NO: 88, and a human IgG1 constant domain. [Invention 1052] A bispecific construct comprising an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody is a. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3 respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6 respectively, or their conservative sequence modifications, b. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, or their conserved sequence modifications, c. Human IgG1 constant domain, comprising (ii) The anti-PD-L1 scFv is, a. Heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, respectively, or their conserved sequence modifications, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively, or their conserved sequence modifications. b. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 35, 36, and 37, respectively, or their conserved sequence modifications, and light chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 38, 39, and 40, respectively, c. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, respectively, or their conservative sequence modifications. d. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively, or their conserved sequence modifications. e. The heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, respectively, or their conservative sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, respectively, or their conservative sequence modifications, f. A bispecific construct comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications. [Invention 1053] A bispecific construct comprising an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody is a. Heavy chain variable region including SEQ ID NO: 17 and light chain variable region including SEQ ID NO: 18, or b. A heavy chain variable region containing Sequence ID 19 and a light chain variable region containing Sequence ID 20, c. Human IgG1 constant domain, comprising (ii) The anti-PD-L1 scFv is, a. Heavy chain variable region including Sequence ID 77 and light chain variable region including Sequence ID 78, b. Heavy chain variable region containing Sequence ID 79 and light chain variable region containing Sequence ID 80, c. Heavy chain variable region including SEQ ID NO. 81 and light chain variable region including SEQ ID NO. 82, d. Heavy chain variable region including SEQ ID NO: 83 and light chain variable region including SEQ ID NO: 84, e. Heavy chain variable region containing Sequence ID 85 and light chain variable region containing Sequence ID 86, or f. A bispecific construct comprising a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88. [Invention 1054] A bispecific construct comprising an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and the human IgG1 constant domain. (ii) A bispecific construct wherein the anti-PD-L1 scFv comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications. [Invention 1055] A bispecific construct comprising an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises a heavy chain variable region including SEQ ID NO: 19, a light chain variable region including SEQ ID NO: 20, and a human IgG1 constant domain. (ii) The anti-PD-L1 scFv is a dual specificity construct comprising a heavy chain variable region including sequence number 83 and a light chain variable region including sequence number 84. [Invention 1056] A bispecific construct comprising an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications, and the human IgG1 constant domain. (ii) A bispecific construct wherein the anti-PD-L1 scFv comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications. [Invention 1057] A bispecific construct comprising an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises a heavy chain variable region including SEQ ID NO: 19, a light chain variable region including SEQ ID NO: 20, and a human IgG1 constant domain. (ii) The anti-PD-L1 scFv is a bispecific construct comprising a heavy chain variable region including sequence number 87 and a light chain variable region including sequence number 88. [Invention 1058] An anti-CD27 antibody or its antigen-binding fragment, wherein the antibody binds to one or more amino acid residues within the range of 85-89 of the extracellular domain (ECD) (SEQ ID NO: 183) of human CD27. [Invention 1059] An anti-CD27 antibody or its antigen-binding fragment, wherein the antibody binds to wild-type human CD27 ECD (SEQ ID NO: 183), but does not bind to mutant forms of the wild-type human CD27 ECD having the following amino acid substitutions: A85S, R87A, N88A, and G89A. [Invention 1060] An anti-CD27 antibody or its antigen-binding fragment, comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6, or their conserved sequence modifications. [Invention 1061] An anti-CD27 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 17 or at least 95% identical thereto, and a light chain variable region containing sequence number 18 or at least 95% identical thereto. [Invention 1062] An anti-CD27 antibody or its antigen-binding fragment, comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications. [Invention 1063] An anti-CD27 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 19 or at least 95% identical thereto, and a light chain variable region containing sequence number 20 or at least 95% identical thereto. [Invention 1064] The anti-CD27 antibody or its antigen-binding fragment has the following characteristics: It induces or enhances the aT cell immune response. b. Block the binding of sCD70 to CD27, c. Equilibrium dissociation constant Kd is 10 -9 M is less than or equal to 10, or the equilibrium dissociation constant Ka is 10 +9 M -1 The above process binds to human CD27. d. Induces specific complement-dependent cytotoxicity (CDC) in cells expressing CD27, It induces antibody-dependent cell-mediated lysis (ADCC) specific to cells expressing e.CD27, f. In combination with a vaccine or endogenous antigen, induce or enhance an antigen-specific immune response in vivo. g. In combination with a vaccine or endogenous antigen, induce or enhance an antigen-specific TH1 immune response in vivo. h. In combination with a vaccine or endogenous antigen, induce or enhance antigen-specific T cell proliferation or activation in vivo, and / or i. An anti-CD27 antibody or antigen-binding fragment of any of the present invention 1058 to 1063 having one or more of the following characteristics: inducing or enhancing T cell activity when combined with simultaneous, separate, or sequential TCR activation. [Invention 1065] A bispecific construct comprising an anti-CD27 antibody according to any of the invention 1058-1063 or its antigen-binding fragment, linked to an anti-PD-L1 binding domain. [Invention 1066] The aforementioned anti-PD-L1 binding domain, a. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, or their conserved sequence modifications, b. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, or their conserved sequence modifications, c. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, or their conserved sequence modifications, d. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications, e. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, or their conserved sequence modifications, respectively, and f. A bispecific construct of the present invention 1064, selected from the group comprising an anti-PD-L1 antibody or its antigen-binding fragment, each comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3, or their conserved sequence modifications, described in SEQ ID NOs. 62, 63, and 64, respectively. [Invention 1067] The aforementioned anti-PD-L1 binding domain, a. Heavy chain variable region including Sequence ID 77 and light chain variable region including Sequence ID 78, b. Heavy chain variable region containing Sequence ID 79 and light chain variable region containing Sequence ID 80, c. Heavy chain variable region including SEQ ID NO. 81 and light chain variable region including SEQ ID NO. 82, d. Heavy chain variable region including SEQ ID NO: 83 and light chain variable region including SEQ ID NO: 84, e. Heavy chain variable region including Sequence ID 85 and light chain variable region including Sequence ID 86, f. A bispecific construct of the present invention 1065 or 1066, selected from the group consisting of a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88. [Invention 1068] A bispecific construct according to any one of the invention 1064 to 1066, wherein the anti-PD-L1 binding domain is scFv. [Invention 1069] An anti-PD-L1 antibody or its antigen-binding fragment, comprising, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, or their conserved sequence modifications. [Invention 1070] An anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 77 or at least 95% identical thereto, and a light chain variable region containing sequence number 78 or at least 95% identical thereto. [Invention 1071] An anti-PD-L1 antibody or its antigen-binding fragment, comprising, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, or their conserved sequence modifications. [Invention 1072] An anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 79 or at least 95% identical thereto, and a light chain variable region containing sequence number 80 or at least 95% identical thereto. [Invention 1073] An anti-PD-L1 antibody or its antigen-binding fragment, comprising, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, or their conserved sequence modifications. [Invention 1074] An anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 81 or at least 95% identical thereto, and a light chain variable region containing sequence number 82 or at least 95% identical thereto. [Invention 1075] An anti-PD-L1 antibody or its antigen-binding fragment, comprising, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications. [Invention 1076] An anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 83 or at least 95% identical thereto, and a light chain variable region containing sequence number 84 or at least 95% identical thereto. [Invention 1077] An anti-PD-L1 antibody or its antigen-binding fragment, comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, or their conserved sequence modifications. [Invention 1078] An anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 85 or at least 95% identical thereto, and a light chain variable region containing sequence number 86 or at least 95% identical thereto. [Invention 1079] An anti-PD-L1 antibody or its antigen-binding fragment, comprising, respectively, the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, or their conserved sequence modifications. [Invention 1080] An anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing sequence number 87 or at least 95% identical thereto, and a light chain variable region containing sequence number 88 or at least 95% identical thereto. [Invention 1081] The anti-PD-L1 antibody or its antigen-binding fragment has the following characteristics: a. Block the binding of PD1 to PD-L1, b. Induces NFAT pathway activation, and / or c. An anti-PD-L1 antibody or antigen-binding fragment of any of the present invention 1069 to 1080 having one or more of the following characteristics: inducing a mixed lymphocyte response. [Invention 1082] A bispecific construct comprising an anti-PD-L1 antigen-binding fragment of any of the present invention 1069-1080, linked to an anti-CD27 binding domain. [Invention 1083] The aforementioned anti-CD27 binding domain a. Anti-CD27 antibodies comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, or their conserved sequence modifications, and b. A bispecific construct of the present invention 1082, selected from the group comprising an anti-CD27 antibody comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3, or their conserved sequence modifications, described in SEQ ID NOs. 10, 11, and 12, respectively. [Invention 1084] The aforementioned anti-CD27 binding domain a. An anti-CD27 antibody comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and b. A bispecific construct of the present invention 1082 or 1083, selected from the group comprising an anti-CD27 antibody comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20. [Invention 1085] A bispecific construct of any of the present invention 1082 to 1084, further comprising the anti-CD27 binding domain and a human IgG1 constant domain. [Invention 1086] A multispecific antibody comprising an anti-PD-L1 antibody having an Fc domain, wherein at least one anti-CD27 antibody-binding domain binds to the Fc domain. [Invention 1087] A multispecific antibody comprising an anti-PD-L1 antibody having an Fc domain, wherein at least one anti-CD27 scFv peptide binds to the Fc domain. [Invention 1088] A multispecific antibody comprising an anti-PD-L1 antibody having an Fc domain, wherein an anti-CD27 scFv peptide is bound to at least one carboxyl terminus of the antibody heavy chain. [Invention 1089] A multispecific antibody comprising an anti-PD-L1 antibody having an Fc domain, wherein an anti-CD27 scFv peptide is bound to at least one carboxyl terminus of the antibody heavy chain, and a further scFv peptide is bound to the carboxyl terminus of the other heavy chain. [Invention 1090] A composition comprising a bispecific construct according to any of Invention 1001-1057, 1065-1068, and 1082-1085, or an antibody or its antigen-binding fragment according to any of Invention 1058-1064 and 1069-1081, or a multispecific antibody according to any of Invention 1086-1089, and a pharmaceutically acceptable carrier. [Invention 1091] A kit comprising a bispecific construct of any of Invention 1001-1057, 1066-1069, and 1082-1085, or an antibody or antigen-binding fragment of any of Invention 1058-1064 and 1069-1081, or a multispecific antibody of any of Invention 1086-1089, or a composition of Invention 1090, along with instructions for use. [Invention 1092] An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody variable region, wherein the antibody variable region comprises the amino acid sequence shown in SEQ ID NOs: 17, 18, 19, 20, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, or 88. [Invention 1093] An isolated nucleic acid molecule containing the nucleotide sequence shown in SEQ ID NOs: 25, 26, 27, 28, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, or 112. [Invention 1094] An isolated nucleic acid molecule comprising nucleotide sequences encoding a heavy chain variable region and a light chain variable region of an antibody, wherein the heavy chain variable region and the light chain variable region each comprise the amino acid sequences shown in SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 77 and 78, SEQ ID NOs: 79 and 80, SEQ ID NOs: 81 and 82, SEQ ID NOs: 83 and 84, SEQ ID NOs: 85 and 86, or SEQ ID NOs: 87 and 88. [Invention 1095] A nucleic acid molecule encoding any of the bispecific constructs 1001-1057, 1065-1068, and 1082-1085 of the present invention. [Invention 1096] A nucleic acid molecule according to any of invention 1092 to 1095 in the form of an expression vector. [Invention 1097] A nucleic acid molecule according to any of the invention 1092 to 1096 in the form of an expression vector, which expresses the aforementioned bispecific construct when administered in vivo to a subject. [Invention 1098] A method for stimulating T cell activity, comprising contacting T cells with any of the bispecific constructs of Invention 1001-1057, 1065-1068, and 1082-1085, or an antibody or antigen-binding fragment of any of Invention 1058-1064 and 1069-1081, or a multispecific antibody of any of Invention 1086-1089, or a composition of Invention 1090. [Invention 1099] A method for inducing or enhancing an immune response in a subject, comprising administering to the subject in an amount effective to induce or enhance an immune response, the bispecific construct of any of Invention 1001-1057, 1065-1068 and 1082-1085, or an antibody or antigen-binding fragment of any of Invention 1058-1064 and 1069-1081, or a multispecific antibody of any of Invention 1086-1089, or a composition of Invention 1090. [Invention 1100] A method for treating a condition or disease in a subject, the method comprising administering to the subject, in an amount effective for treating the condition or disease, a bispecific construct of any of Invention 1001-1057, 1065-1068 and 1082-1085, or an antibody or antigen-binding fragment of any of Invention 1058-1064 and 1069-1081, or a multispecific antibody of any of Invention 1086-1089, or a composition of Invention 1088. [Invention 1101] A method for treating a condition or disease in a subject, wherein the method comprises administering an anti-CD27 antibody or its antigen-binding fragment in combination with an anti-PD-L1 antibody or its antigen-binding fragment to the subject. (i) The anti-CD27 antibody or its antigen-binding fragment is a. An anti-CD27 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6, or their conserved sequence modifications, respectively, and b. Selected from the group consisting of an anti-CD27 antibody or its antigen-binding fragment, each comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3, or their conserved sequence modifications, respectively, as described in SEQ ID NOs. 10, 11, and 12, (ii) The anti-PD-L1 antibody or its antigen-binding fragment is a. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, or their conserved sequence modifications, b. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, or their conserved sequence modifications, c. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, or their conserved sequence modifications, d. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, or their conserved sequence modifications, e. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, or their conserved sequence modifications, respectively, and f. A method selected from the group comprising an anti-PD-L1 antibody or its antigen-binding fragment, each comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, or their conserved sequence modifications, and the light chain variable regions CDR1, CDR2, and CDR3, or their conserved sequence modifications, described in SEQ ID NOs. 62, 63, and 64, respectively. [Invention 1102] A method for treating a condition or disease in a subject, wherein the method comprises administering an anti-CD27 antibody or its antigen-binding fragment in combination with an anti-PD-L1 antibody or its antigen-binding fragment to the subject. (i) The anti-CD27 antibody or its antigen-binding fragment is a. An anti-CD27 antibody or its antigen-binding fragment comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and b. Selected from the group consisting of an anti-CD27 antibody or its antigen-binding fragment comprising SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20 or the sequence, (ii) The anti-PD-L1 antibody or its antigen-binding fragment is a. An anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region containing SEQ ID NO: 77 and a light chain variable region containing SEQ ID NO: 78. b. An anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80. c. An anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82. d. An anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84. e. An anti-PD-L1 antibody or its antigen-binding fragment comprising a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86, and f. A method selected from the group comprising an anti-PD-L1 antibody or its antigen-binding fragment, comprising a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88. [Invention 1103] The method according to the present invention 1101 or 1102, wherein the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are administered separately. [Invention 1104] The method according to the present invention 1101 or 1102, wherein the anti-CD27 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are administered together. [Invention 1105] The method according to any one of the present invention 1098 to 1104, wherein the subject is suffering from a condition or disease in which stimulation of an immune response is desired. [Invention 1106] The method of the present invention 1105, wherein the aforementioned condition or disease is cancer. [Invention 1107] The method of the present invention 1106, wherein the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and glioblastoma. [Invention 1108] It is a bispecific tetravalent antibody, i) Two IgG heavy chains, ii) Two light chains, iii) comprising two single-stranded Fv(scFv) domains, A bispecific tetravalent antibody in which the two IgG heavy chains and two light chains form an IgG moiety that specifically binds to human PD-L1, the two scFv domains each specifically bind to human CD27, and each scFv domain binds to one C-terminal residue of the IgG heavy chain via a connector sequence. [Invention 1109] The bispecific tetravalent antibody of the present invention 1108, wherein the IgG heavy chain is an IgG1 heavy chain. [Invention 1110] A bispecific tetravalent antibody according to Invention 1108 or Invention 1109, wherein the light chain is a kappa light chain. [Invention 1111] Each scFv domain has one of the following structural sequences: i) N-terminal variable heavy chain domain - linker - variable light chain domain - C-terminus, or ii) N-terminal variable light chain domain - linker - variable heavy chain domain - C-terminus, and in each case, the linker is (G4S) m A bispecific tetravalent antibody according to any of the present inventions 1108 to 1110, comprising the amino acid sequence and having m as at least an integer of 3. [Invention 1112] A bispecific tetravalent antibody according to any of the present invention 1108 to 1111, wherein the connector sequence includes the amino acid sequence of G4S. [Invention 1113] A bispecific tetravalent antibody according to any of the present invention 1108 to 1112, wherein the connector sequence includes the amino acid sequence GS2G4S. [Invention 1114] A bispecific tetravalent antibody according to any of the present invention 1108 to 1113, wherein the linker sequence includes the amino acid sequence of (G4S)4. [Invention 1115] A bispecific tetravalent antibody according to any of the present invention 1108 to 1114, wherein at least one of the anti-CD27 scFv domains comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NOs. 7, 8, and 9, or their conserved sequence modifications, and light chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NOs. 10, 11, and 12, or their conserved sequence modifications. [Invention 1116] A bispecific tetravalent antibody according to any of the Invention 1108 to 1115, wherein at least one of the anti-PD-L1 IgG heavy chains comprises heavy chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NOs. 59, 60, and 61, respectively, or their conserved sequence modifications, and at least one of the anti-PD-L1 light chains comprises light chain CDR1, CDR2, and CDR3 domains having the sequences described in SEQ ID NOs. 62, 63, and 64, respectively, or their conserved sequence modifications. [Invention 1117] A composition comprising a bispecific tetravalent antibody according to any of Invention 1108 to 1116 and a pharmaceutically acceptable carrier. [Invention 1118] A kit comprising a bispecific tetravalent antibody according to any of Invention 1108 to 1116 or a composition according to Invention 1117, and instructions for use. [Invention 1119] A method for stimulating T cell activity, comprising contacting T cells with a bispecific tetravalent antibody according to any of Invention 1108 to 1116 or a composition according to Invention 1117. [Invention 1120] A method for inducing or enhancing an immune response in a subject, comprising administering to the subject a bispecific tetravalent antibody according to any of Invention 1108 to 1116 or a composition according to Invention 1117 in an amount effective for inducing or enhancing an immune response in the subject. [Invention 1121] A method for treating a condition or disease in a subject, the method comprising administering to the subject a bispecific tetravalent antibody according to any of Invention 1108 to 1116 or a composition according to Invention 1117 in an amount effective for treating the condition or disease. [Invention 1122] The method according to any one of the present invention 1119 to 1121, wherein the subject is suffering from a condition or disease in which stimulation of an immune response is desired. [Invention 1123] The method of the present invention 1122, wherein the aforementioned condition or disease is cancer. [Invention 1124] The method of the present invention 1123, wherein the cancer is selected from the group consisting of colorectal cancer, ovarian cancer, renal cell carcinoma, head and neck squamous cell carcinoma, and glioblastoma. [Brief explanation of the drawing]

[0089] [Figure 1] This graph shows the binding of CD27 antibodies 2B3 and 3C2 to recombinant human CD27 as a function of antibody concentration. [Figure 2] This graph shows the binding of CD27 antibodies 2B3 and 3C2 to recombinant cynomolgus monkey CD27. [Figure 3] This graph shows the high level of binding of CD27 antibodies 2B3 and 3C2 to Ramos cells that express CD27 on their surface. [Figure 4] This graph shows the high levels of CD27 antibodies 2B3 and 3C2 binding to T cells. [Figure 5] This graph shows that antibodies 2B3 and 3C2 significantly block the binding of CD70 to CD27. [Figure 6] This graph shows, as a function of antibody concentration, that antibodies 2B3 and 3C2 induced significant NFκB activation. [Figure 7] This graph shows that antibodies 2B3 and 3C2 increase T cell proliferation. [Figure 8] This graph shows the binding of anti-PD-L1 antibodies to human PD-L1 as a function of antibody concentration. [Figure 9] This graph shows the binding of anti-PD-L1 antibodies to recombinant cynomolgus monkey PD-L1. [Figure 10] This graph shows, as a function of antibody concentration, that an anti-PD-L1 antibody significantly blocks the binding of PD-L1 to PD1. [Figure 11] This graph shows the high level of binding of anti-PD-L1 antibodies to human PD-L1-expressing cells as a function of antibody concentration. [Figure 12] This graph shows the high level of binding of anti-PD-L1 antibodies to human dendritic cells as a function of antibody concentration. [Figure 13] This graph shows that anti-PD-L1 antibodies block intercellular PD1 / PD-L1 interactions, leading to activation of the NFAT pathway. [Figure 14] This graph shows that anti-PD-L1 antibodies induce a mixed lymphocyte response as a function of antibody concentration. [Figure 15A] This is a representative DNA expression vector containing an anti-CD27 light chain, an anti-CD27 heavy chain, and a c-terminal anti-PD-L1 single-strand (VL+VH) peptide. [Figure 15B] This is a diagram of a CD27 / PD-L1 bispecific antibody protein in which an anti-PD-L1 antibody is linked to an anti-CD27 scFv. [Figure 15C] This is a diagram of a CD27 / PD-L1 bispecific antibody protein in which an anti-CD27 antibody is linked to an anti-PD-L1 scFv. [Figure 15D] This is a table of representative anti-CD27 / anti-PD-L1 bispecific constructs. [Figure 15E] This paper describes the characterization of the bispecific antibody CDX-527 by HPLC and gel electrophoresis. [Figure 16] This graph shows the binding of anti-CD27 / anti-PD-L1 bispecific constructs (BsAb) to CD27 and PD-L1 using a bifunctional ELISA. [Figure 17] This graph shows the increased NFκB activation induced by the anti-CD27 / anti-PD-L1 bispecific construct compared to antibody 1F5 or 2B3 alone. [Figure 18] A graph showing that an anti-CD27 / anti-PD-L1 bispecific construct blocks the PD1 / PD-L1 interaction and induces NFAT pathway activation as a function of antibody concentration. [Figure 19] A graph showing that an anti-CD27 / anti-PD-L1 bispecific construct increases IL-2 production / secretion in the mixed lymphocyte reaction compared to antibody AbX (a known anti-PD-L1 monoclonal antibody), 8B1, or 9H9 alone. [Figure 20] Figures 20A and 20B are graphs showing that an anti-CD27 / anti-PD-L1 bispecific construct (e.g., CD27xAbX) induces a higher CD8 T cell response compared to a CD27 monoclonal antibody alone. [Figure 21] A Kaplan-Meier curve showing improved survival of mice treated with a bispecific construct (e.g., CD27xAbX) compared to CD27 and PD-L1 antibodies administered alone or in combination in a mouse tumor model. [Figure 22A] A graph showing a decrease in tumor weight in mice treated with a bispecific construct (e.g., CD27xAbX) compared to CD27 and PD-L1 antibodies administered alone or in combination. [Figure 22B] A graph showing an increase in the percentage of CD8 T cells in mice treated with a bispecific construct (e.g., CD27xAbX) compared to CD27 and PD-L1 antibodies administered alone or in combination. [Figure 22C] A graph showing an increase in the percentage of CD4 T cells in mice treated with a bispecific construct (e.g., CD27xAbX) compared to CD27 and PD-L1 antibodies administered alone or in combination. [Figure 22D] A graph showing an increase in activated CD8 T cells in mice treated with a bispecific construct (e.g., CD27xAbX) compared to CD27 and PD-L1 antibodies administered alone or in combination. [Figure 23]Figures 23A and 23B are graphs showing that anti-CD27 Ab (e.g., CDX-1127) upregulates PD-L1 expression in tumor cells and tumor-infiltrating cells. [Figure 24] This graph shows the binding of the anti-CD27 / anti-PD-L1 bispecific construct CDX-527 to CD27 and PD-L1, as measured by a bifunctional ELISA. [Figure 25] This graph shows the increased NFκB activation induced by the anti-CD27 / anti-PD-L1 bispecific construct CDX-527 compared to antibody 1F5 or 2B3 alone (left graph) and in the presence of soluble FcγR1 (right graph). [Figure 26] This graph shows the increased IL-2 production / secretion in mixed lymphocyte reactions induced by the anti-CD27 / anti-PD-L1 bispecific construct CDX-527 compared to antibodies 2B3 or 9H9 alone or in combination. [Figure 27] This graph shows the increased intracellular IL-2 production / secretion by the anti-CD27 / anti-PD-L1 bispecific construct CDX-527 compared to the antibody 2B3 and 9H9 combination. [Figure 28] This graph shows the serum levels of CDX-527 in NHP pharmacokinetic studies. [Figure 29] This graph shows the increased blockade of PD-1 signaling with the 9H9x2B3 configuration compared to the 2B3x9H9 configuration. [Figure 30] This graph shows the increased T cell activation with the 9H9x2B3 configuration compared to the 2B3x9H9 configuration. [Figure 31] This graph shows the increased stimulation of the vaccine-induced CD8+ T cell response by AbXx2B3 compared to 2B3xAbX. [Figure 32] This graph shows the increased antitumor activity of AbXx2B3 compared to 2B3xAbX. [Figure 33] This graph shows the blockage of PD-L1 binding to CD80 by the anti-PD-L1 antibodies AbX and 9H9. [Figure 34]Figure 34 (SEQ ID NO: 183) shows the sequence of the extracellular domain (ECD) of wild-type huCD27, as well as a mutant with substitutions at positions 85, 87, 88, and 89. [Figure 35] This graph shows the binding of anti-CD27 antibody 2B3 to both wild-type huCD27 and mutant huCD27 having the sequence shown in Figure 34. [Figure 36] This graph shows the improved production (expression) of the modified 9H9x2B3(DD) construct compared to the original (unmodified) 9H9x2B3 construct. [Modes for carrying out the invention]

[0090] Detailed description of the invention CD27 is an important co-stimulating receptor that can be used in immunotherapy using agonist molecules. CD27 plays a crucial role in various immunological processes, including T cell survival, activation, and effector function, as well as natural killer (NK) cell proliferation and cytotoxic activity. These events occur in response to the appropriate interaction between the ligand (CD70) and CD27, resulting in intracellular signaling events that lead to NF-κB activation and the expression of related genes. As with most co-stimulating molecules, co-stimulation through the T cell receptor (TCR) is also required for T cells to be effectively stimulated by either the ligand or the agonist antibody.

[0091] Most CD27 agonist molecules require multimerization or crosslinking for their activity. For example, anti-CD27 antibodies that are agonists are crosslinked through the interaction of their Fc domains with Fc receptors via cis or trans interactions. In vitro, this can also be done artificially using a secondary anti-Ig antibody or by adsorbing the antibody onto a solid-phase matrix. The need for FcR interaction in anti-CD27 agonist antibodies means that the T cell stimulating activity is at least partially dependent on the number of FcR-expressing cells.

[0092] To eliminate the need for FcR interaction, CD27 agonists have been invented that either 1) eliminate the need for interaction with receptors other than CD27 (referred to herein as CD27 supercrosslinkers), or 2) provide crosslinking through interaction with alternating receptors that may be different specific cell types that do not express FcR (these alternating receptors (referred to herein as CD27 multispecific agents) may also provide additional functions).

[0093] In one embodiment, the present invention provides a bispecific construct (or multispecific construct) comprising an anti-CD27 antibody or its antigen-binding fragment linked to an anti-PD-L1 antibody or its antigen-binding fragment. Herein, such an anti-CD27 × anti-PD-L1 bispecific agent of the present invention has been shown to exhibit synergistic effects in vivo, such as enhancement of immune parameters and inhibition of tumor growth, compared to the co-administration of an anti-CD27 antibody and an anti-PD-L1 antibody (see Examples 9 and 10).

[0094] To make the present invention easier to understand, certain terms are defined first. Additional definitions are provided throughout the embodiments for carrying out the invention.

[0095] A.Definition As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject having an immune disorder. The term “non-human animal” includes all vertebrates, such as non-human primates, mammals and non-mammals such as sheep, dogs, cattle, chickens, amphibians, and reptiles.

[0096] As used herein, the term "binding domain" refers to a portion of a protein or antibody that contains amino acid residues that interact with an antigen. Binding domains include, but are not limited to, antibodies (e.g., full-length antibodies), as well as antigen-binding portions thereof. A binding domain confers specificity and affinity for an antigen to a binder. The term also encompasses any protein having a binding domain that is homologous or mostly homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources or may be produced partially or wholly synthetically.

[0097] As referred to herein, the term "antibody" includes the whole antibody and any antigen-binding fragment (i.e., "antigen-binding portion") or single-chain form thereof. In a preferred embodiment, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is composed of a heavy-chain variable region (abbreviated herein as V H and a heavy-chain constant region. The heavy-chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light-chain variable region (abbreviated herein as V L and a light-chain constant region. The light-chain constant region is composed of one domain, CL. V H and V L regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). The CDRs specified herein are those according to the Kabat system, unless otherwise indicated. Each V H and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0098] As used herein, the term “antigen-binding fragment” (or simply “antibody fragment”) of an antibody refers to one or more fragments or portions of an antibody that possess the ability to specifically bind to an antigen (e.g., human CD27). Such “fragments” may be, for example, about 8 to about 1500 amino acids long, preferably about 8 to about 745 amino acids long, preferably about 8 to about 300, for example, about 8 to about 200 amino acids long, or about 10 to about 50 or 100 amino acids long. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding fragment” of an antibody include (i)V L , V H (ii) a monovalent fragment consisting of CL and CH1 domains, a Fab fragment, (ii) a bivalent fragment F(ab')2 fragment containing two Fab fragments linked by disulfide crosslinks in the hinge region, (iii) V Fd fragment consisting of H and CH1 domains, (iv) single arm of antibody V L and V H Fv fragment consisting of domains, (v)V H The dAb fragment consists of domains (Ward et al., (1989) Nature 341:544-546), and (vi) an isolated complementarity-determining region (CDR), or (vii) a combination of two or more isolated CDRs that can be joined by a synthetic linker. Furthermore, the two domains of the Fv fragment, V L and V HThese are encoded by separate genes, but they are V L Region and V H The regions may be joined using recombination techniques by a synthetic linker that allows them to be constructed as a single protein chain forming a monovalent molecule (known as single-stranded Fv (sFv); see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-stranded antibodies are also intended to be included in the term "antigen-binding fragment" of antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. The antigen-binding portion can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0099] As used herein, the term “monoclonal antibody” refers to an antibody that exhibits a single binding specificity and affinity for a particular epitope. Therefore, the term “human monoclonal antibody” refers to an antibody that exhibits a single binding specificity and has a variable region and an optional constant region derived from a human germline immunoglobulin sequence. In one embodiment, a human monoclonal antibody is produced by a hybridoma containing B cells having a genome containing human heavy chain and light chain transgenes fused to immortalized cells, obtained from a transgenic non-human animal, such as a transgenic mouse.

[0100] As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal with respect to human immunoglobulin genes, or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express the antibody, for example, from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, for example, during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region contains antigen-binding domains encoded by various genes that are rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region may be further modified by multiple single-amino acid changes (referred to as somatic mutations or hypermutations) to increase the affinity of antibodies to foreign antigens. The constant region changes in a further response to the antigen (i.e., isotype switching). Thus, rearranged and somatically mutated nucleic acid molecules encoding light-chain and heavy-chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecule, but instead may be substantially identical or similar (i.e., have at least 80% identity).

[0101] The term "human antibody" includes antibodies having variable and constant regions (if present) of a human germline immunoglobulin sequence. The human antibodies of the present invention may also include amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo) (see Lonberg, N. et al. (1994) Nature 368(6474):856-859, Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101, Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. NYAcad. Sci 764:536-546). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been transplanted onto a human framework sequence (i.e., chimeric and humanized antibodies).

[0102] As used herein, “isolated antibody” is intended to refer to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to human CD27 substantially does not contain antibodies that specifically bind to antigens other than human CD27, and an isolated antibody that specifically binds to human PD-L1 substantially does not contain antibodies that specifically bind to antigens other than human PD-L1). However, isolated antibodies that specifically bind to an epitope may cross-react to the same antigen from different species. In addition, isolated antibodies typically substantially do not contain other cellular material and / or chemicals.

[0103] The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from both adjacent or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial structure. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which antigen-derived duplicate or adjacent peptides (e.g., CD27 or PD-L1) are tested for reactivity with a given antibody (e.g., anti-CD27 or anti-PD-L1 antibody). Methods for determining the spatial structure of an epitope include techniques described in the relevant art and herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0104] The term "antibody that binds to the same epitope" as another antibody is intended to encompass antibodies that interact with, i.e., bind to, the same structural region as the reference anti-CD27 antibody on human CD27. The "same epitope" to which the antibody binds may be a linear epitope or a conformational epitope formed by the tertiary folding of the antigen.

[0105] The term "competitive antibody" refers to an antibody that competes with a reference anti-CD27 antibody for binding to human CD27, that is, an antibody that competitively inhibits the binding of the reference anti-CD27 antibody to CD27. A "competitive antibody" may bind to the same CD27 epitope as the reference anti-CD27 antibody, to a duplicate epitope, or sterically interfere with the binding of the reference anti-CD27 antibody to CD27.

[0106] Antibodies that recognize or compete for the same epitope can be identified using conventional techniques. Such techniques include, for example, immunoassays that demonstrate the ability of one antibody to block the binding of another antibody to its target antigen, i.e., competitive binding assays. Competitive binding is determined by assays in which the immunoglobulin being tested inhibits the specific binding of the reference antibody to a common antigen, such as CD27. Numerous types of competitive binding assays, e.g., solid-phase direct or indirect radioimmunoassays (RIAs), solid-phase direct or indirect enzyme immunoassays (EIAs), sandwich competitive assays (see Stahli et al., Methods in Enzymology 9:242 (1983)), solid-phase direct biotin-avidin EIA (see Kirkland et al., J.Immunol. 137:3614 (1986)), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)), solid-phase direct labeling RIA using I-125 labeling (see Morel et al., Mol.Immunol. 25(1):7 (1988)), solid-phase direct biotin-avidin EIA (see Cheung et al., Virology 176:546 (1990) and directly labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)) are known. Typically, such assays involve the use of purified antigen, unlabeled test immunoglobulin, and labeled reference immunoglobulin bound to a solid surface or cell having one of these. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of test immunoglobulin. Usually, test immunoglobulin is present in excess. Usually, when a competitive antibody is present in excess, it inhibits the specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, and 70-75% or more.

[0107] Other techniques include epitope mapping methods, such as X-ray analysis of antigen:antibody complex crystals, which provide atomic resolution for epitopes. Other methods monitor the binding of antibodies to antigen fragments or variants of antigens, where loss of binding due to modification of amino acid residues within the antigen sequence is often considered an indicator of the epitope component. In addition, computational combinatorial methods can also be used for epitope mapping. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from a combinatorial phage-presenting peptide library. The peptides are then considered leads for defining the epitopes corresponding to the antibodies used to screen the peptide library. Computational algorithms have also been developed for epitope mapping that have been shown to map discontinuous epitopes in three-dimensional structures.

[0108] As used herein, the terms “specific binding,” “selective binding,” “selectively binding,” and “specifically binding” refer to antibodies that bind to an epitope on a given antigen. Typically, antibodies are measured by surface plasmon resonance (SPR) technique on a BIACORE2000 instrument (for example, using recombinant human CD27 as the analyte and the antibody as the ligand) to approximately 10 -8 M, 10 -9 M or 10 -10 Approximately 10, such as less than M or less. -7 Equilibrium dissociation constant (K) less than M D It binds to a given antigen with an affinity at least twice as great as its affinity to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). The terms “antigen-recognizing antibody” and “antigen-specific antibody” are used interchangeably herein with the term “antigen-specific antibody.”

[0109] When used herein, "K DThe term is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the human antibodies of the present invention are determined by surface plasmon resonance (SPR) technique using a BIACORE2000 instrument (e.g., using recombinant human CD27 as the analyte and the antibody as the ligand), and are 10 -9 M or 10 -10 Approximately 10, such as less than M or less. -8 Dissociation equilibrium constants (K) less than or equal to M D ) and combine with CD27.

[0110] As used herein, the term "kd" is intended to refer to the off-rate constant for the dissociation of an antibody from an antibody / antigen complex.

[0111] As used herein, the term "ka" is intended to refer to the rate constant of association between an antibody and an antigen.

[0112] As used herein, the term "EC50" refers to the concentration of an antibody or its antigen-binding moiety that elicits 50% of the maximum response, i.e., a response midway between the maximum response and the baseline, in either an in vitro or in vivo assay.

[0113] As used herein, “isotype” refers to the antibody class (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. In one embodiment, the human monoclonal antibody of the present invention is of the IgG1 isotype. In another embodiment, the human monoclonal antibody of the present invention is of the IgG2 isotype.

[0114] As used herein, the terms “inhibit” and “block” (for example, referring to the inhibition / blockage of the binding of CD70 to CD27 and / or PD1 to PD-L1) are used interchangeably and encompass both partial and complete inhibition / blockage. Inhibition / blockage preferably reduces or alters the normal level or type of activity that occurs when binding occurs without inhibition or blockage. Inhibition and blockage are also intended to include any measurable decrease in the binding affinity of CD70 when in contact with an anti-CD27 antibody compared to CD70 not in contact with an anti-CD27 antibody, e.g., inhibition of CD70 binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the anti-CD27 antibody inhibits the binding of CD70 by at least about 70%. In another embodiment, the anti-CD27 antibody inhibits the binding of CD70 by at least 80%. Inhibition and blockade are also intended to include any measurable decrease in the binding affinity of PD1 when in contact with the anti-PD-L1 antibody compared to PD1 not in contact with the anti-PD-L1 antibody, e.g., inhibiting the binding of PD1 by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the anti-PD-L1 antibody inhibits the binding of PD1 by at least about 70%. In another embodiment, the anti-PD-L1 antibody inhibits the binding of PD1 by at least 80%.

[0115] As used herein, the term “cross-reactivity” refers to the ability of the anti-CD27 binding domain or anti-PD-L1 binding domain of the present invention to bind to CD27 or PD-L1 from different species, respectively. For example, the CD27 binding domain of the present invention that binds to human CD27 may also bind to CD27 of another species. Similarly, the anti-PD-L1 binding domain of the present invention that binds to human PD-L1 may also bind to PD-L1 of another species. As used herein, cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR, ELISA), or by detecting binding to cells that physiologically express CD27 or by other means of functional interaction. Methods for determining cross-reactivity include, for example, standard binding assays described herein by Biacore® surface plasmon resonance (SPR) analysis using a Biacore® 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or by flow cytometry techniques.

[0116] As used herein, the term “naturally occurring” as applied to a substance refers to the fact that the substance can be found in nature. For example, polypeptides or polynucleotide sequences present in living organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified by humans in a laboratory are naturally occurring.

[0117] The present invention also encompasses “conservative sequence modifications” of any of the sequences described in SEQ ID NOs: 1 to 160, i.e., nucleotide and amino acid sequence modifications that do not inhibit the binding of VH and VL sequences, which are encoded by a nucleotide sequence for the antigen or contain an amino acid sequence for the antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into SEQ ID NOs: 1 to 160 by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues predicted in anti-CD27 antibodies are preferably replaced with other amino acid residues from the same side chain family. Methods for identifying conserved nucleotide and amino acid substitutions that do not exclude antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993), Kobayashi et al. Protein Eng. 12(10):879-884 (1999), and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0118] In certain embodiments, a conserved amino acid sequence modification refers to up to one, two, three, four, or five conserved amino acid substitutions in a CDR sequence as described herein. For example, each such CDR may contain up to five conserved amino acid substitutions, e.g., up to four (i.e., less than or equal to) conserved amino acid substitutions, e.g., up to three (i.e., less than or equal to) conserved amino acid substitutions, e.g., up to two (i.e., less than or equal to) conserved amino acid substitutions, or one or fewer conserved amino acid substitutions.

[0119] Alternatively, in another embodiment, mutations can be randomly introduced along all or part of the anti-CD27 or anti-PD-L1 binding domain coding sequence by means of saturation mutagenesis, and the resulting modified anti-CD27 or anti-PD-L1 antibody can be screened for binding activity.

[0120] In the case of nucleic acids, the term "substantial homology" indicates that, when optimally aligned and compared, two nucleic acids or their specified sequences are identical in at least about 80%, typically at least about 90%–95%, and more preferably at least about 98%–99.5%, of nucleotides, including appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists if the segments hybridize to the complement of the chain under selective hybridization conditions.

[0121] In the case of amino acids, the term "substantial homology" indicates that, when optimally aligned and compared, two amino acid sequences or their specified sequences are identical in at least about 80%, typically at least about 90% to 95%, and more preferably at least about 98% to 99% or 99.5%, of the amino acids, including appropriate amino acid insertions or deletions.

[0122] The percentage of identity between two arrays is a function of the number of identical positions shared by the arrays, taking into account the number of gaps that need to be introduced for optimal alignment of the two arrays and the length of each gap (i.e., identity % = number of identical positions / total number of positions × 100). As illustrated in the non-restrictive examples below, comparing arrays and determining the percentage of identity between two arrays can be achieved using mathematical algorithms.

[0123] The percentage of identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix, as well as 40, 50, 60, 70, or 80 gap weights and 1, 2, 3, 4, 5, or 6 length weights. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, 12 gap length penalties, and 4 gap penalties. In addition, the degree of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)) algorithm, which is incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, as well as gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0124] The nucleic acid and protein sequences of the present invention can be further used, for example, as "query sequences" to perform searches against public databases to identify relevant sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) described in Altschul, et al. (1990) J.Mol.Biol.215:403-10. A BLAST nucleotide search can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain a nucleotide sequence identical to that of the nucleic acid molecule of the present invention. A BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain an amino acid sequence identical to that of the protein molecule of the present invention. To obtain gapped alignment for comparison purposes, gapped BLAST may be used as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. Please refer to http: / / www.ncbi.nlm.nih.gov.

[0125] B. Anti-CD27 antibodies and their binding domains Novel anti-CD27 antibodies and their binding domains are provided herein. “CD27” (also referred to as “CD27 molecule,” “CD27L receptor,” “S1521,” “T cell activating antigen CD27,” “TNFRSF7,” “MGC20393,” “tumor necrosis factor receptor superfamily, member 7,” “T cell activating antigen S152,” “Tp55,” “tumor necrosis factor receptor superfamily member 7,” “CD27 antigen,” and “T cell activating antigen CD27”) refers to a receptor that is a member of the TNF receptor superfamily and binds to ligand CD70. CD27 is essential for the generation and long-term maintenance of T cell immunity and plays a crucial role in the regulation of B cell activation and immunoglobulin synthesis. The term “CD27” includes any variant or isoform of CD27 naturally expressed by cells (e.g., human CD27 deposited with GENBANK® with accession number AAH12160.1, as described in SEQ ID NO: 173). Therefore, the CD27 binding domains of the present invention may cross-react with CD27 from non-human species. Alternatively, the CD27-binding domain may be specific to human CD27 and not exhibit cross-reactivity with other species. CD27 or any variants and isoforms thereof may be isolated from cells or tissues that naturally express them, or they may be recombinantly produced using techniques well known in the art and / or techniques described herein. Preferably, the CD27-binding domain targets human CD27 having a normal glycosylation pattern.

[0126] "CD70" (also referred to as "CD70 molecule," "CD27L," "CD27LG," "TNFSF7," "tumor necrosis factor (ligand) superfamily member 7," "CD27 ligand," "CD70 antigen," "surface antigen CD70," "tumor necrosis factor ligand superfamily, member 7," "Ki-24 antigen," and "CD27-L") refers to the ligand for CD27 (see, for example, Bowman MR et al., J.Immunol. 1994 Feb 15;152(4):1756-61). CD70 is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) ligand family. It is a surface antigen on activated T and B lymphocytes that induces the proliferation of co-stimulated T cells, enhances the generation of cytotoxic T cells, and contributes to T cell activation. CD70 has also been suggested to play a role in B cell activation, the cytotoxic function of natural killer cells, and the regulation of immunoglobulin synthesis (Hintzen RQ et al., J.Immunol. 1994 Feb 15;152(4):1762-73). Genbank® accession number NP_001243 reports the amino acid sequence of human CD70 (SEQ ID NO: 174).

[0127] An exemplary anti-CD27 antibody is antibody 3C2 as described herein. In one embodiment, the anti-CD27 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 3C2. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 3C2 having the sequence described in SEQ ID NO: 17, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 3C2 having the sequence described in SEQ ID NO: 18. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains having the sequences described in SEQ ID NOs: 1, 2 and 3, respectively, or their conserved sequence modifications, and the light chain CDR1, CDR2 and CDR3 domains having the sequences described in SEQ ID NOs: 4, 5 and 6, respectively, or their conserved sequence modifications. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 17. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 18. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0128] Another exemplary anti-CD27 antibody is antibody 2B3 as described herein. In one embodiment, the anti-CD27 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 2B3. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 2B3 having the sequence described in SEQ ID NO: 19, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 3C2 having the sequence described in SEQ ID NO: 20. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 7, 8 and 9, respectively, and the light chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 10, 11 and 12, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 19. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 20. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

[0129] Sequences substantially identical to the anti-C27 binding domains described herein (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences described herein) are also provided. In one embodiment, the anti-CD27 binding domain includes a heavy chain variable region containing sequence number 17, sequence number 19, or a sequence that is at least 90% identical to them (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences described herein). In another embodiment, the anti-CD27 binding domain includes a light chain variable region containing sequence number 18, sequence number 20, or a sequence that is at least 90% identical to them (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences described herein). In another embodiment, the anti-CD27 binding domain includes a heavy chain variable region containing the sequence of sequence number 17 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of sequence number 18 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-CD27 binding domain includes a heavy chain variable region containing the sequence of sequence number 19 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of sequence number 20 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0130] Anti-CD27 antibodies or their binding domains that compete for binding with any of the anti-CD27 antibodies or their binding domains described herein, or that bind to the same epitope as any of the anti-CD27 antibodies or their binding domains described herein, are also suitable for use and are provided herein. For example, in one embodiment, an anti-CD27 antibody or its binding domain competes with antibody 3C2 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 3C2) for binding to CD27. In another embodiment, an anti-CD27 antibody or its binding domain competes with antibody 2B3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 2B3) for binding to CD27. In another embodiment, an antibody or its anti-CD27 binding domain binds to the same epitope on CD27 as antibody 3C2 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 3C2). In another embodiment, the anti-CD27 antibody or its binding domain binds to the same CD27 epitope as antibody 2B3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 2B3).

[0131] In one embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises a heavy chain variable region containing a sequence of SEQ ID NO: 17 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 18 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0132] In another embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively. In another embodiment, the anti-CD27 antibody or its antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NOs: 19 or a sequence at least 95% identical thereto, and a light chain variable region containing SEQ ID NOs: 20 or a sequence at least 95% identical thereto.

[0133] In another embodiment, the anti-CD27 antibody or its antigen-binding fragment has the following functional characteristics: it induces or enhances a T cell-mediated immune response; it blocks (e.g., partially or completely) the binding of sCD70 to CD27; it induces NFκB activation; it increases T cell proliferation; and it has an equilibrium dissociation constant Kd of 10. -9 M is less than or equal to 10, or the equilibrium association constant Ka is 10 +9 M -1 The above describes a functional characteristic which is one or more of the following: binding to human CD27; inducing specific complement-mediated cytotoxicity (CDC) in CD27-expressing cells; inducing antibody-dependent cell-mediated cytotoxicity (ADCC) specific lysis in CD27-expressing cells; inducing or enhancing an antigen-specific immune response in vivo in combination with a vaccine or endogenous antigen; inducing or enhancing an antigen-specific TH1 immune response in vivo in combination with a vaccine or endogenous antigen; inducing or enhancing antigen-specific T cell proliferation or activation in vivo in combination with a vaccine or endogenous antigen; and / or inducing or enhancing T cell activity when combined with simultaneous, separate, or sequential TCR activation.

[0134] C. Anti-PD-L1 antibody and binding domain Novel anti-PD-L1 antibodies and their binding domains are provided herein. Where used herein, the terms “Programmed Cell Death 1 Ligand 1,” “PD-L1,” “PDCD1 Ligand 1,” “Programmed Death Ligand 1,” “B7 Homogeno 1,” “B7-H1,” and “CD274” are used interchangeably and include variants, isoforms, species homologs of human PD-L1, and analogs having at least one common epitope with PD-L1. The complete PD-L1 sequence can be found in GenBank accession number NP_001254635, as described in Sequence ID No. 176.

[0135] Programmed cell death ligand 1 (PD-L1) is a 40 kDa type 1 transmembrane protein that is presumed to play a major role in suppressing the immune system during certain events such as pregnancy, tissue allografting, autoimmune diseases, and other medical conditions (such as hepatitis). Normally, the immune system responds to exogenous antigens associated with exogenous or endogenous danger signals, which trigger the proliferation of antigen-specific CD8+ T cells and / or CD4+ helper cells. Binding of PD-L1 to PD-1 transmits an inhibitory signal that reduces the proliferation of these T cells and can also induce apoptosis, which is further mediated by lower regulation of the gene Bcl-2. Where used herein, the terms “programmed cell death 1,” “programmed cell death 1,” “protein PD-1,” “PD-1,” “PD1,” “PDCD1,” “hPD-1,” and “hPD-I” are used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The complete PD-1 sequence can be found in GenBank accession number NP_005009, as described in SEQ ID NO: 175.

[0136] PD-L1 is present in large quantities in various human cancers (Dong et al. (2002) Nat. Med. 8:787-9). The interaction between PD-1 and PD-L1 leads to a decrease in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J. Mol. Med. 81:281-7, Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314, Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). By inhibiting the local interaction between PD-1 and PD-L1, immunosuppression can be reversed, and if the interaction between PD-1 and PD-L2 is similarly blocked, this effect is additive (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7, Brown et al. (2003) J. Immunol. 170:1257-66).

[0137] An exemplary anti-PD-L1 antibody is antibody 7H7 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 7H7. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 7H7 having the sequence described in SEQ ID NO: 77, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 7H7 having the sequence described in SEQ ID NO: 78. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 29, 30 and 31, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 32, 33 and 34, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 77. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 77. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 77 and SEQ ID NO: 78, respectively.

[0138] Another exemplary anti-PD-L1 antibody is antibody 1B3 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 1B3. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 1B3 having the sequence described in SEQ ID NO: 79, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 1B3 having the sequence described in SEQ ID NO: 80. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 35, 36 and 37, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 38, 39 and 40, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 79. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 80. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 79 and SEQ ID NO: 80, respectively.

[0139] Another exemplary anti-PD-L1 antibody is antibody 3B6 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 3B6. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 3B6 having the sequence described in SEQ ID NO: 81, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 3B6 having the sequence described in SEQ ID NO: 82. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 41, 42 and 43, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 44, 45 and 46, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 81. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 82. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 81 and SEQ ID NO: 82, respectively.

[0140] Another exemplary anti-PD-L1 antibody is antibody 8B1 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 8B1. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 8B1 having the sequence described in SEQ ID NO: 83, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 8B1 having the sequence described in SEQ ID NO: 84. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 47, 48 and 49, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 50, 51 and 52, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 83. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 84. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 83 and SEQ ID NO: 84, respectively.

[0141] Another exemplary anti-PD-L1 antibody is antibody 4A3 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 4A3. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 4A3 having the sequence described in SEQ ID NO: 85, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 4A3 having the sequence described in SEQ ID NO: 86. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 53, 54 and 55, respectively, and the light chain CDR1, CDR2 and CDR3 domains, or their conserved sequence modifications, having the sequences described in SEQ ID NOs: 56, 57 and 58, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 85. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 86. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 85 and SEQ ID NO: 86, respectively.

[0142] Another exemplary anti-PD-L1 antibody is antibody 9H9 as described herein. In one embodiment, the anti-PD-L1 antibody or its binding domain includes the heavy chain and light chain CDR or variable region of antibody 9H9. In another embodiment, the antibody or its binding domain includes the CDR1, CDR2 and CDR3 domains of the heavy chain variable region of antibody 9H9 having the sequence described in SEQ ID NO: 87, and the CDR1, CDR2 and CDR3 domains of the light chain variable region of antibody 9H9 having the sequence described in SEQ ID NO: 88. In another embodiment, the antibody or its binding domain includes the heavy chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 59, 60 and 61, respectively, and the light chain CDR1, CDR2 and CDR3 domains or their conserved sequence modifications having the sequences described in SEQ ID NOs: 62, 63 and 64, respectively. In another embodiment, the antibody or its binding domain includes the heavy chain variable region having the amino acid sequence described in SEQ ID NO: 87. In another embodiment, the antibody or its binding domain includes the light chain variable region having the amino acid sequence described in SEQ ID NO: 88. In another embodiment, the antibody or its binding domain includes heavy chain and light chain variable regions having the amino acid sequences described in SEQ ID NO: 87 and SEQ ID NO: 88, respectively.

[0143] The antibody sequence may also be the consensus sequence of several antibodies. For example, in one embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region CDR1 containing an amino acid sequence selected from the consensus sequence: (T,S)(S,Y,H)WMS (SEQ ID NO: 167). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region CDR2 containing SEQ ID NO: 168. In yet another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region CDR3 containing SEQ ID NO: 169. In yet another embodiment, the anti-PD-L1 binding domain includes a light chain variable region CDR1 containing SEQ ID NO: 170. In yet another embodiment, the anti-PD-L1 binding domain includes a light chain variable region CDR2 containing SEQ ID NO: 171. In yet another embodiment, the anti-PD-L1 binding domain includes a light chain variable region CDR3 containing SEQ ID NO: 172.

[0144] Sequences substantially identical to (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to) the anti-PD-L1 antibodies and their binding domains described herein are also included by the present invention. In one embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, or sequences at least 90% identical to them (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences described herein). In another embodiment, the anti-PD-L1 binding domain includes a light chain variable region containing sequence SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, or sequences that are at least 90% identical to them (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequences). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 77 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 78 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing the sequence of SEQ ID NO: 79 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 80 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 81 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 82 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 83 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 84 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 85 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 86 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 binding domain includes a heavy chain variable region containing sequence number 87 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 88 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0145] Anti-PD-L1 antibodies and their binding domains that compete for binding with any of the anti-PD-L1 antibodies or their binding domains described herein, or that bind to the same epitope as any of the anti-PD-L1 antibodies or their binding domains described herein, are also suitable for use and are provided herein. For example, in one embodiment, an anti-PD-L1 antibody or its binding domain competes with antibody 7H7 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7H7) for binding to PD-L1. In another embodiment, an anti-PD-L1 antibody or its binding domain binds to the same epitope on PD-L1 as antibody 7H7 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 7H7).

[0146] In another embodiment, the anti-PD-L1 antibody or its binding domain competes with antibody 1B3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 1B3) for binding to PD-L1. In another embodiment, the anti-PD-L1 antibody or its binding domain binds to the same epitope on PD-L1 as antibody 1B3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 1B3).

[0147] In another embodiment, the anti-PD-L1 antibody or its binding domain competes with antibody 3B6 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 3B6) for binding to PD-L1. In another embodiment, the anti-PD-L1 antibody or its binding domain binds to the same epitope on PD-L1 as antibody 3B6 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 3B6).

[0148] In another embodiment, the anti-PD-L1 antibody or its binding domain competes with antibody 8B1 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 8B1) for binding to PD-L1. In another embodiment, the anti-PD-L1 antibody or its binding domain binds to the same epitope on PD-L1 as antibody 8B1 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 8B1).

[0149] In another embodiment, the anti-PD-L1 antibody or its binding domain competes with antibody 4A3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 4A3) for binding to PD-L1. In another embodiment, the anti-PD-L1 antibody or its binding domain binds to the same epitope on PD-L1 as antibody 4A3 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 4A3).

[0150] In another embodiment, the anti-PD-L1 antibody or its binding domain competes with antibody 9H9 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 9H9) for binding to PD-L1. In another embodiment, the anti-PD-L1 antibody or its binding domain binds to the same epitope on PD-L1 as antibody 9H9 (or an antibody having heavy and light chain CDRs and / or heavy and light chain variable region sequences corresponding to antibody 9H9).

[0151] In another embodiment, the anti-PD-L1 binding domain is an anti-PD-L1 antibody or its antigen-binding portion. In one embodiment, the anti-PD-L1 antibody or its antigen-binding fragment comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing the sequence of SEQ ID NO: 77 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 78 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 38, 39, and 40, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 79 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 80 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 41, 42, and 43, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 44, 45, and 46, respectively.In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 81 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 82 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 50, 51, and 52, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 83 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 84 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 53, 54, and 55, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 56, 57, and 58, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment includes a heavy chain variable region containing a sequence of SEQ ID NO: 85 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO: 86 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively. In another embodiment, the anti-PD-L1 antibody or its antigen-binding fragment comprises a heavy chain variable region containing a sequence of SEQ ID NO. 87 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing a sequence of SEQ ID NO. 88 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0152] In another embodiment, an anti-PD-L1 antibody or its antigen-binding fragment has one or more of the following functional features: (a) blocking the binding of PD1 to PD-L1 (e.g., partially or completely), (b) inducing NFAT pathway activation, and / or (c) inducing a mixed lymphocyte response.

[0153] D. Bispecific constructs A bispecific construct comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain is provided herein. Such bispecific constructs are also provided, which can be linked to one or more additional binders to form a multispecific construct.

[0154] A "bispecific" or "bifunctional" construct is an artificial hybrid having two different binding domain (e.g., heavy / light chain) pairs and two different binding sites. Bispecific constructs can be generated by various methods, including hybridoma fusion or Fab' fragment linking. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990) and Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

[0155] As used herein, the term “linked” refers to the association of two or more molecules. Linking may be covalent or non-covalent. Linking may also be genetic (i.e., recombinant fusion). Such linking can be achieved using a variety of techniques recognized in the art, such as chemical coupling and recombinant protein production.

[0156] In the case of chemical conjugation, suitable reagents and methods for coupling two or more moieties, particularly two or more antibodies or their fragments, together are known in the art. Various coupling or crosslinking agents are commercially available and can be used to conjugate anti-CD27 binding domains and anti-PD-L1 binding domains. Non-limiting examples include sulfo-SMCC, protein A, carbodiimide, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Sulfo-SMCC, SPDP, and DTNB are preferred agents, with sulfo-SMCC being particularly preferred. Other suitable procedures for crosslinking components (e.g., binding domains) with crosslinking agents are known in the art. See, for example, Karpovsky, B. et al., (1984) J. Exp. Med. 160:1686, Liu, M. et al., (1985) Proc. Natl. Acad. Sci USA 82:8648, Segal, D. and Perez, P., U.S. Patent No. 4,676,980, and Brennan, M. (1986) Biotechniques 4:424.

[0157] In the case of genetic manipulation, a nucleic acid molecule encoding an anti-CD27 binding domain can be inserted into a suitable expression vector using standard recombinant DNA techniques. Nucleic acid molecules encoding an anti-PD-L1 binding domain can also be inserted into the same expression vector, resulting in operable linkage to the CD27 binding domain (e.g., in-frame cloning), thereby yielding an expression vector encoding a fusion protein that is a bispecific construct. Preferably, the anti-PD-L1 binding domain is operable linkage to the C-terminal region of the heavy chain of the anti-CD27 binding domain. Other suitable expression vectors and cloning strategies for preparing the bispecific constructs described herein are known in the art.

[0158] To express a bispecific construct in host cells, the coding region of the binding domain is combined with a cloning promoter sequence, a leader sequence, a translation initiation sequence, another leader sequence, a constant region sequence, a 3' untranslated sequence, a polyadenylation sequence, and a transcription termination sequence to form an expression vector construct. These constructs can be used to express, for example, full-length human IgG1κ or IgG4κ antibodies. Full-human, humanized, and chimeric antibodies used in the bispecific constructs described herein also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar plasmids can be constructed to express other heavy chain isotypes or antibodies containing lambda light chains.

[0159] After preparing an expression vector encoding a bispecific construct, the bispecific construct can be recombinantly expressed in host cells using standard transfection methods. For example, in one embodiment, the nucleic acid encoding the bispecific construct can be ligated to an expression vector such as a eukaryotic expression plasmid, such as those used in the GS gene expression system disclosed in WO87 / 04462, WO89 / 01036 and EP338841, or other expression systems well known in the art. A purified plasmid containing the cloned bispecific construct gene can be introduced into eukaryotic host cells such as CHO cells or NSO cells, or into other eukaryotic cells such as plant-derived cells, fungal or yeast cells. Methods used to introduce these genes may be methods described in the art, such as electroporation, lipofectin, lipofectamine, or others. After introducing the expression vector into host cells, cells expressing the bispecific construct can be identified and selected. These cells represent transfectomas that can then be amplified for their expression levels and upscaled to produce the bispecific construct. Alternatively, these clonal bispecific constructs may be expressed in other expression systems or complete organisms such as E. coli, or synthetically. Recombinant bispecific constructs can be isolated and purified from these culture supernatants and / or cells.

[0160] The bispecific constructs of the present invention, whether prepared by chemical coupling or genetic engineering, can be isolated and purified using one or more methodologies for protein purification that are well established in the art. Preferred methods for isolation and purification include, but are not limited to, gel filtration chromatography, affinity chromatography, and anion exchange chromatography. A particularly preferred method is, for example, gel filtration chromatography using a Superdex 200 column. The isolated and purified bispecific constructs can be evaluated using standard methods such as SDS-PAGE analysis.

[0161] Therefore, in one embodiment, the anti-PD-L1 binding domain and the anti-CD27 binding domain are genetically fused. In another embodiment, the anti-PD-L1 binding domain and the anti-CD27 binding domain are chemically coupled. In one embodiment, the anti-PD-L1 binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-CD27 binding domain is ligated to the C-terminus of the heavy chain of the anti-PD-L1 binding domain. In another embodiment, the anti-CD27 binding domain is scFv. In another embodiment, the anti-CD27 binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-PD-L1 binding domain is ligated to the C-terminus of the heavy chain of the anti-CD27 binding domain. In another embodiment, the anti-PD-L1 binding domain is scFv.

[0162] Exemplary bispecific constructs are shown in Tables 1 and 2 below, where the binding domain is defined by a CDR sequence (Table 1) or a variable region sequence (Table 2).

[0163] (Table 1) Exemplary bispecific constructs (CDRs) TIFF0007839817000001.tif162170

[0164] (Table 2) Exemplary bispecific constructs (VRs) TIFF0007839817000002.tif73170

[0165] In one embodiment, a bispecific construct comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain, (i) The anti-CD27 binding domain is a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 4, 5, and 6, respectively, or b. comprising heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively. (ii) The anti-PD-L1 binding domain is a. Heavy chain variable region CDR1 containing an amino acid sequence selected from the consensus sequence (T,S)(S,Y,H)WMS (SEQ ID NO: 167), b. Heavy chain variable region CDR2 including sequence number 168, c. Heavy chain variable region CDR3 including sequence number 169, d. Light chain variable region CDR1 including sequence number 170, e. Light chain variable region CDR2 including sequence number 171, A bispecific construct is provided that includes a light chain variable region CDR3 containing sequence number 172.

[0166] In another embodiment, a bispecific construct comprising an anti-CD27 binding domain linked to an anti-PD-L1 binding domain, (i) The anti-CD27 binding domain is a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 4, 5, and 6, respectively, or b. comprising heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively. (ii) The anti-PD-L1 binding domain is a. A heavy chain variable region containing sequence number 77 or a sequence identical to it by at least 95% (for example, at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 78 or a sequence identical to it by at least 95% (for example, at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence), b. A heavy chain variable region containing sequence number 79 or a sequence identical to it by at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 80 or a sequence identical to it by at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 9% identical to the aforementioned sequence), c. A heavy chain variable region containing sequence number 81 or a sequence identical to it by at least 95% (for example, at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 82 or a sequence identical to it by at least 95% (for example, at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence), d. A heavy chain variable region containing sequence number 83 or a sequence identical to it by at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 84 or a sequence identical to it by at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence), e. A heavy chain variable region containing sequence number 85 or a sequence identical to it by at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 86 or a sequence identical to it by at least 95% (e.g., at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence), or f. A bispecific construct is provided, comprising a heavy chain variable region containing sequence number 87 or at least 95% identical thereto (e.g., at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing sequence number 88 or at least 95% identical thereto (e.g., at least 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0167] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 29, 30, and 31, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 32, 33, and 34, respectively.

[0168] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region comprising SEQ ID NO: 77 and a light chain variable region comprising SEQ ID NO: 78.

[0169] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 38, 39, and 40, respectively.

[0170] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80.

[0171] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 41, 42, and 43, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 44, 45, and 46, respectively.

[0172] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82.

[0173] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 47, 48, and 49, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 50, 51, and 52, respectively.

[0174] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region comprising SEQ ID NO: 83 and a light chain variable region comprising SEQ ID NO: 84.

[0175] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 53, 54, and 55, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 56, 57, and 58, respectively.

[0176] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86.

[0177] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively, and an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 59, 60, and 61, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 62, 63, and 64, respectively.

[0178] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 17 and a light chain variable region containing SEQ ID NO: 18, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0179] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively.

[0180] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 77 and a light chain variable region containing SEQ ID NO: 78.

[0181] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, and an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, respectively.

[0182] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 79 and a light chain variable region containing SEQ ID NO: 80.

[0183] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively, and an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 41, 42, and 43, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 44, 45, and 46, respectively.

[0184] In another embodiment, the bispecific construct includes (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 81 and a light chain variable region containing SEQ ID NO: 82.

[0185] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively.

[0186] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84.

[0187] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, respectively.

[0188] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 85 and a light chain variable region containing SEQ ID NO: 86.

[0189] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively, and (b) an anti-PD-L1 binding domain comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 59, 60, and 61, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 62, 63, and 64, respectively.

[0190] In another embodiment, the bispecific construct comprises (a) an anti-CD27 binding domain comprising a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20, and (b) an anti-PD-L1 binding domain comprising a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0191] In certain embodiments, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv. (i) Anti-CD27 scFv is, a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 4, 5, and 6, respectively, or b. comprising heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively. (ii) Anti-PD-L1 antibodies are a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 29, 30, and 31, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 32, 33, and 34, respectively. b. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 35, 36, and 37, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 38, 39, and 40, respectively. c. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 41, 42, and 43, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 44, 45, and 46, respectively. d. Heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 50, 51, and 52, respectively. e. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 53, 54, and 55, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 56, 57, and 58, respectively, or f. Heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively. g. Contains the human IgG1 constant domain.

[0192] In another specific embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) Anti-CD27 antibody, a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 4, 5, and 6, respectively, or b. Heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 10, 11, and 12, respectively, c. Includes the human IgG1 constant domain, (ii) Anti-PD-L1 scFv is, a. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 29, 30, and 31, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 32, 33, and 34, respectively. b. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 35, 36, and 37, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 38, 39, and 40, respectively. c. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 41, 42, and 43, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 44, 45, and 46, respectively. d. Heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 50, 51, and 52, respectively. e. Heavy chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 53, 54, and 55, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in Sequence IDs 56, 57, and 58, respectively, or f. Includes heavy chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 59, 60, and 61, respectively, and light chain variable regions CDR1, CDR2, and CDR3 as described in SEQ ID NOs. 62, 63, and 64, respectively.

[0193] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv is, a. Heavy chain variable region including SEQ ID NO: 17 and light chain variable region including SEQ ID NO: 18, or b. Includes a heavy chain variable region containing Sequence ID 19 and a light chain variable region containing Sequence ID 20, (ii) Anti-PD-L1 antibodies are a. Heavy chain variable region including Sequence ID 77 and light chain variable region including Sequence ID 78, b. Heavy chain variable region containing Sequence ID 79 and light chain variable region containing Sequence ID 80, c. Heavy chain variable region including SEQ ID NO. 81 and light chain variable region including SEQ ID NO. 82, d. Heavy chain variable region including SEQ ID NO: 83 and light chain variable region including SEQ ID NO: 84, e. Heavy chain variable region containing Sequence ID 85 and light chain variable region containing Sequence ID 86, or f. A heavy chain variable region including sequence number 87 and a light chain variable region including sequence number 88, g. Contains the human IgG1 constant domain.

[0194] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) Anti-CD27 antibody, a. Heavy chain variable region including SEQ ID NO: 17 and light chain variable region including SEQ ID NO: 18, or b. A heavy chain variable region containing Sequence ID 19 and a light chain variable region containing Sequence ID 20, c. Includes the human IgG1 constant domain, (ii) Anti-PD-L1 scFv is, a. Heavy chain variable region including Sequence ID 77 and light chain variable region including Sequence ID 78, b. Heavy chain variable region containing Sequence ID 79 and light chain variable region containing Sequence ID 80, c. Heavy chain variable region including SEQ ID NO. 81 and light chain variable region including SEQ ID NO. 82, d. Heavy chain variable region including SEQ ID NO: 83 and light chain variable region including SEQ ID NO: 84, e. Heavy chain variable region containing Sequence ID 85 and light chain variable region containing Sequence ID 86, or f. Includes a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0195] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 10, 11, and 12, respectively. (ii) The PD-L1 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively, and the human IgG1 constant domain.

[0196] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively, and the human IgG1 constant domain. (ii) Anti-PD-L1 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively.

[0197] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20 (ii) The anti-PD-L1 antibody comprises a heavy chain variable region containing SEQ ID NO: 83, a light chain variable region containing SEQ ID NO: 84, and a human IgG1 constant domain.

[0198] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises a heavy chain variable region containing SEQ ID NO: 19, a light chain variable region containing SEQ ID NO: 20, and a human IgG1 constant domain. (ii) The anti-PD-L1 scFv comprises a heavy chain variable region containing SEQ ID NO: 83 and a light chain variable region containing SEQ ID NO: 84.

[0199] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 10, 11, and 12, respectively. (ii) The anti-PD-L1 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively, and the human IgG1 constant domain.

[0200] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, respectively, the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively, and the human IgG1 constant domain. (ii) Anti-PD-L1 scFv comprises heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively.

[0201] In another embodiment, the bispecific construct comprises an anti-PD-L1 antibody linked to anti-CD27 scFv, (i) Anti-CD27 scFv includes a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20 (ii) The anti-PD-L1 antibody comprises a heavy chain variable region containing SEQ ID NO: 87, a light chain variable region containing SEQ ID NO: 88, and a human IgG1 constant domain.

[0202] In another embodiment, the bispecific construct comprises an anti-CD27 antibody linked to anti-PD-L1 scFv, (i) The anti-CD27 antibody comprises a heavy chain variable region containing SEQ ID NO: 19, a light chain variable region containing SEQ ID NO: 20, and a human IgG1 constant domain. (ii) The anti-PD-L1 scFv comprises a heavy chain variable region containing SEQ ID NO: 87 and a light chain variable region containing SEQ ID NO: 88.

[0203] In another embodiment, a bispecific construct is provided, comprising one of the anti-CD27 antibodies or antigen-binding fragments described herein, ligated to an anti-PD-L1 binding domain. In one embodiment, the anti-PD-L1 binding domain is a. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively. b. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, respectively. c. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 41, 42, and 43, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 44, 45, and 46, respectively. d. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 47, 48, and 49, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 50, 51, and 52, respectively. e. An anti-PD-L1 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 53, 54, and 55, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 56, 57, and 58, respectively, and f. Selected from the group consisting of an anti-PD-L1 antibody or its antigen-binding fragment, comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 59, 60, and 61, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 62, 63, and 64, respectively.

[0204] In another embodiment, the anti-PD-L1 binding domain is selected from the group consisting of (a) a heavy chain variable region including SEQ ID NO: 77 and a light chain variable region including SEQ ID NO: 78, (b) a heavy chain variable region including SEQ ID NO: 79 and a light chain variable region including SEQ ID NO: 80, (c) a heavy chain variable region including SEQ ID NO: 81 and a light chain variable region including SEQ ID NO: 82, (d) a heavy chain variable region including SEQ ID NO: 83 and a light chain variable region including SEQ ID NO: 84, (e) a heavy chain variable region including SEQ ID NO: 85 and a light chain variable region including SEQ ID NO: 86, and (f) a heavy chain variable region including SEQ ID NO: 87 and a light chain variable region including SEQ ID NO: 88. In a particular embodiment, the anti-PD-L1 binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-PD-L1 binding domain is scFv.

[0205] In another embodiment, a bispecific construct is provided, comprising one of the anti-PD-L1 antibodies described herein or their antigen-binding fragments, ligated to an anti-CD27 binding domain. In one embodiment, the anti-CD27 binding domain comprises an anti-CD27 antibody comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 1, 2, and 3, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 4, 5, and 6, respectively. In another embodiment, the anti-CD27 binding domain comprises an antibody comprising a heavy chain variable region containing the sequence of SEQ ID NO: 17 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 18 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In another embodiment, the anti-CD27 binding domain comprises an anti-CD27 antibody comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 7, 8, and 9, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs: 10, 11, and 12, respectively. In another embodiment, the anti-C27 binding domain comprises a CD27 antibody comprising a heavy chain variable region containing the sequence of SEQ ID NO: 19 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence) and a light chain variable region containing the sequence of SEQ ID NO: 20 or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence). In one embodiment, the anti-CD27 binding domain is scFv. In another embodiment, the anti-CD27 binding domain further comprises a human IgG1 constant domain.

[0206] In another embodiment, the bispecific construct has one or more of the following functional features: inducing NFκB activation, increasing T cell proliferation, inducing a CD8 T cell response, and / or increasing IL-2 production. In another embodiment, the bispecific construct increases IL-2 production by at least about 1.5 times (e.g., at least 2 times, 2.5 times, 3 times, 3.5 times, or 4 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone. In another embodiment, the bispecific construct induces a CD8 T cell response at least about 2 times greater (e.g., at least 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8.0 times, 8.5 times, or 9 times) than anti-CD27 monoclonal antibody alone. In another embodiment, the bispecific construct increases survival by at least approximately 1.5 times (e.g., at least 1.5 times, 2.0 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone or in combination. In another embodiment, the bispecific construct reduces tumor weight by at least approximately 1.5 times (e.g., at least 1.5 times, 2.0 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, or 5 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone or in combination. In another embodiment, the bispecific construct increases T cell production by at least about 1.5 times (e.g., at least 1.5 times, 2.0 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8.0 times, 8.5 times, or 9 times) compared to anti-CD27 monoclonal antibody or anti-PD-L1 monoclonal antibody alone or in combination.

[0207] In certain embodiments, the bispecific constructs described herein exhibit synergistic effects (e.g., in enhancing the immune response in vivo) compared to the use of a combination of anti-CD27 binding domains and anti-PD-L1 binding domains (i.e., co-administration of unbound antibodies).

[0208] E. Composition Compositions comprising, for example, one or a combination of the binding domains, antibodies, or antigen-binding fragments, bispecific constructs, or multispecific constructs described herein, formulated together with a carrier (e.g., a pharmaceutically acceptable carrier), are also provided herein.

[0209] As used herein, the terms “carrier” and “pharmaceutically acceptable carrier” include any physiologically compatible solvent, salt, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., any of the binding domains, antibodies, or their antigen-binding fragments, bispecific constructs, or multispecific constructs described herein) may be coated with materials to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0210] Examples of adjuvants that can be used with the binding domains, antibodies, or their antigen-binding fragments, bispecific constructs, or multispecific constructs described herein include Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, Mich.), Merck adjuvant 65 (Merck and Company, Inc., Rahway, NJ), and AS-2 (SmithKline). This includes, but is not limited to, Beecham (Philadelphia, Pa.), aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate, salts of calcium, iron or zinc, insoluble suspensions of acylated tyrosine, acylated sugars, cationic or anionic derivatized polysaccharides, polyphosphazenes, biodegradable microspheres, cytokines such as GM-CSF, interleukin-2, -7, -12 and other similar factors, 3D-MPL, CpG oligonucleotides, and monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A.

[0211] MPL adjuvants are available from Corixa Corporation (Seattle, Wash; see, for example, U.S. Patents 4,436,727, 4,877,611, 4,866,034 and 4,912,094). CpG-containing oligonucleotides (with unmethylated CpG dinucleotides) are well known and are described, for example, in WO96 / 02555, WO99 / 33488 and U.S. Patents 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.

[0212] Further alternative adjuvants include, for example, saponins such as Quil A, or its derivatives including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), estine, digitonin, or Gypsophila or Chenopodium quinoa saponins, Montanide ISA720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), and adjuvants from the SBAS series (e.g., SmithKline). Polyoxyethylene ether adjuvants such as SBAS-2 or SBAS-4 (available from Beecham, Rixensart, Belgium), Detox (Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.), and other aminoalkylglucosaminide 4-phosphates (AGPs), as described in WO99 / 52549A1, imiquimod [S-26308, R-837] (Harrison, et al., Vaccine 19:1820-1826, 2001) and reximod [S-28463, R-848] (Vasilakos, et al., Cellular Synthetic imidazoquinolines such as immunology204:64-74,2000, and tucaresol, which are carbonyl and amine Schiff bases constitutively expressed on the surface of antigen-presenting cells and T cells (Rhodes, J. et al.).(Nature 377:71-75, 1995), for example, pro-inflammatory cytokines such as interferon, GM-CSF, IL-1 alpha, IL-1 beta, TGF-alpha and TGF-beta; Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15, IL-18 and IL-21; Th2 inducers such as IL-4, IL-5, IL-6, IL-10 and IL-13; as well as other chemokines and co-stimulating genes such as MCP-1, MIP-1 alpha, MIP-1 beta, RANTES, TCA-3, CD80, CD86 and CD40L; cytokines as proteins or peptides; ligand-targeting immunostimulants such as CTLA-4 and L-selectin; apoptosis-stimulating proteins and peptides such as Fas; Vaxfectin (Reyes et al., Vaccine These include synthetic lipid adjuvants such as squalene, alpha-tocopherol, polysorbate 80, DOPC, and cholesterol (19:3778-3786, 2001), endotoxins, [LPS] (Beutler, B., Current Opinion in Microbiology 3:23-30, 2000), ligands that trigger the Toll receptor and produce Th1-inducing cytokines such as synthetic mycobacterial lipoproteins, mycobacterial protein p19, peptidoglycan, teichoic acid, and lipid A, as well as CT (cholera toxin, subunits A and B) and LT (thermally unstable enterotoxin from E. coli, subunits A and B), heat shock protein family (HSP), and LLO (listeriolisin O, WO01 / 72329). These and various other Toll-like receptor (TLR) agonists are described, for example, by Kanzler et al., Nature Medicine, May 2007, Vol. 13, No. 5.

[0213] A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound without conferring any undesirable toxicological effects (see, for example, Berge, SM, et al (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0214] The compositions of the present invention can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. The active compound can be prepared using a carrier that protects the compound from rapid release, such as sustained-release formulations including implantable tablets, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0215] To administer the compounds of the present invention via a specific route of administration, it may be necessary to coat the compounds with a material to prevent their inactivation, or to administer the compounds together with the material. For example, the compounds may be administered to a subject in a suitable carrier, such as liposomes, or in a diluent. Acceptable diluents include physiological saline and buffered aqueous solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

[0216] The carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such culture media and agents for pharmaceutically active substances is known in the art. Unless any conventional culture media or agent is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is intended. Complementary active compounds may also be incorporated into the compositions.

[0217] Therapeutic compositions typically must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other regular structures suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol and sorbitol, or sodium chloride in the composition. Extended absorption of the injectable composition can be achieved by including absorption-delaying agents, such as monostearate salts and gelatin, in the composition.

[0218] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in a suitable solvent, along with one or a combination of the components listed above as needed, and then microfiltration for sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed above. For sterile powders used to prepare sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield the active ingredient powder in addition to any additional desired components from its previously sterile-filtered solution.

[0219] The medication plan is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, whether a single bolus is administered, or several divided doses are administered over time, the dose may be proportionally reduced or increased as indicated by an emergency in the treatment situation. For example, the antibody of the present invention may be administered once or twice a week by subcutaneous or intramuscular injection, or once or twice a month by subcutaneous or intramuscular injection.

[0220] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage forms. As used herein, unit dosage forms refer to physically distinct units suitable as a single dose for the subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms of the present invention are indicated and depend directly on (a) the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art to compound such active compounds for the treatment of the sensitivity of an individual.

[0221] Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0222] In the case of therapeutic compositions, the formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations can be conveniently presented in unit dosage forms and can be prepared by any method known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single-dose dosage form will vary depending on the target being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single-dose dosage form is generally the amount of the composition that produces the therapeutic effect. Generally, out of 100 percent, this amount is in the range of about 0.001 percent to about 90 percent, preferably about 0.005 percent to about 70 percent, and most preferably about 0.01 percent to about 30 percent of the active ingredient.

[0223] Formulations of the present invention suitable for intravaginal administration include vaginal suppositories, tampons, creams, gels, pastes, foams, or spray formulations containing carriers known to be suitable in the art. Dosage forms of the compositions of the present invention for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as needed.

[0224] As used herein, the terms “parenteral administration” and “administered parenterally” mean a mode of administration other than enteral and local administration, usually by injection, and do not limit themselves to intravenous, intramuscular, intra-arterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0225] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0226] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured both by the sterilization procedures described above and by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, extended absorption of injectable dosage forms can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0227] When the compounds of the present invention are administered to humans and animals as pharmaceuticals, they can be given alone or as pharmaceutical compositions containing, for example, 0.001 to 90% (more preferably 0.005 to 70%, such as 0.01 to 30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0228] Regardless of the selected route of administration, the compounds of the present invention, and / or the pharmaceutical compositions of the present invention, which can be used in a preferred hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0229] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention or its esters, salts, or amides used, the route of administration, administration time, excretion rate, duration of treatment of the particular compound used, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and prior medical history of the patient being treated, and similar factors well known in the field of medicine. A physician or veterinarian skilled in the art can easily determine and prescribe the required effective amount of pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the compound of the present invention used in the pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Generally, a preferred daily dose of the composition of the present invention is the amount of the compound that is the minimum effective dose to produce a therapeutic effect. Such an effective dose will generally depend on the factors mentioned above. Administration is preferably intravenous, intramuscular, intraperitoneal, or subcutaneous, and preferably proximal to the target site. If necessary, the effective daily dose of the therapeutic composition may be administered as 2, 3, 4, 5, 6 or more partial doses, optionally administered separately in unit dosage forms, at appropriate intervals throughout the day. While it is possible to administer the compounds of the present invention alone, it is preferable to administer the compounds as a pharmaceutical formulation (composition).

[0230] The therapeutic composition can be administered together with medical devices known in the art. For example, in a preferred embodiment, the therapeutic composition of the present invention can be administered by a needle-free subcutaneous injection device such as the devices disclosed in U.S. Patents 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 or 4,596,556. Examples of well-known implantable tablets and modules useful in the present invention include U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194 disclosing a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233 disclosing a drug infusion pump for delivering drugs at a precise infusion rate; U.S. Patent No. 4,447,224 disclosing a variable flow rate implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196 disclosing an osmotic drug delivery system having multiple chamber compartments; and U.S. Patent No. 4,475,196 disclosing an osmotic drug delivery system. Many other such implantable tablets, delivery systems, and modules are known to those skilled in the art.

[0231] In certain embodiments, the antibodies of the present invention may be formulated to ensure appropriate distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention (if necessary) cross the BBB, they may be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patents 4,522,811, 5,374,548 and 5,399,331. Liposomes contain one or more portions that are selectively transported to specific cells or organs, and can thus enhance targeted drug delivery (see, for example, VVRanade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, for example, U.S. Patent No. 5,416,016 by Low et al.), mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PGBloeman et al., (1995) FEBS Lett. 357:140, M. Owais et al., (1995) Antimicrob. Agents Chemother. 39:180), surfactants, protein A receptor (Briscoe et al., (1995) Am. J. Physiol. 1233:134), p120 (Schreier et al.), and different species of which may comprise components of the formulations and molecules of the present invention. This includes al. (1994) J. Biol. Chem. 269:9090), and also see K. Keinanen; M. Laukkanen (1994) FEBS Lett. 346:123, and J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273. In one embodiment of the present invention, the therapeutic compound of the present invention is formulated in liposomes, and in a more preferred embodiment, the liposomes include a targeting moiety. In the most preferred embodiment, the therapeutic compound in the liposomes is delivered to a proximal site of the tumor or infection by bolus injection. The composition should be fluid enough to allow for easy injection.It must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi.

[0232] The ability of a compound to inhibit cancer can be evaluated in animal model systems that predict efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the compound's inhibitory ability (in vitro inhibition by assays known to those skilled in the art). A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate the symptoms of the target. Those skilled in the art will be able to determine such an amount based on factors such as the size of the target, the severity of the symptoms of the target, and the specific composition or route of administration selected.

[0233] The composition must be sterile and fluid enough to be deliverable by syringe. In addition to water, the carrier may be isotonic buffered saline, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid ethylene glycol), or suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of a surfactant. Often, it is preferable to include isotonic agents, such as sugars, polyhydric alcohols such as mannitol or sorbitol, and sodium chloride in the composition. Extended absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate or gelatin, in the composition.

[0234] As described above, when the active compound is suitably protected, the compound can be administered orally, for example, with an inert diluent or an assimilated food carrier.

[0235] F. Nucleic acids As used herein, the term “nucleic acid molecule” is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0236] A binding domain, antibody, or antibody moiety that binds to CD27 and / or PD-L1 (e.g., V H , V L When used herein in relation to nucleic acids encoding CD27 and / or PD-L1, the term “isolated nucleic acid molecule” is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the binding domain, antibody, or antibody moiety do not include other nucleotide sequences encoding the binding domain, antibody, or antibody moiety that bind to antigens other than CD27 and / or PD-L1, and in which these other sequences may naturally be adjacent to the nucleic acids of human genomic DNA.

[0237] Nucleic acids can exist in whole cells, cell lysates, or in partially purified or substantially pure forms. Nucleic acids are “isolated” or “substantially purified” when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques including alkali / SDS treatment, CsCl band formation, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0238] The nucleic acid molecules of the present invention, derived from cDNA, genomes, or mixtures thereof, are often natural sequences (except for modification restriction sites, etc.), but can be mutated according to standard techniques to provide gene sequences. In the case of coding sequences, these mutations can affect the amino acid sequence, if desired. In particular, DNA sequences that are substantially identical to or derived from the natural V, D, J sequences, constant sequences, switch sequences, and other such sequences described herein are intended (where “derived” indicates that the sequence is identical or modified from another sequence).

[0239] Nucleic acids are "operably ligated" or "operatably ligated" when they are in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operationally ligated to a coding sequence if it affects the transcription of that sequence. With respect to transcriptional regulatory sequences, operational ligation means that the ligated DNA sequences are adjacent and, if necessary, ligate two adjacent and read-frame-dependent protein-coding regions. In the case of switch sequences, operational ligation indicates that the sequence can result in switch recombination.

[0240] Isolated nucleic acid molecules, bispecific constructs and multispecific constructs encoding the binding domains, antibodies, or their antigen-binding moieties described herein, as well as expression vectors containing such nucleic acids and host cells containing such expression vectors. In another embodiment, nucleic acid molecules, bispecific constructs or multispecific constructs encoding any of the binding domains, antibodies, or their antigen-binding moieties described herein are provided. In another embodiment, the nucleic acid molecule is in the form of an expression vector. In another embodiment, the nucleic acid molecule is in the form of an expression vector, bispecific construct or multispecific construct that, when administered in vivo to a subject, expresses the binding domains, antibodies, or their antigen-binding moieties.

[0241] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody variable region, the antibody variable region comprising an amino acid sequence shown in SEQ ID NOs: 17, 18, 19, 20, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, or an amino acid sequence that is at least 90% identical thereto (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the aforementioned sequences). In another embodiment, the nucleic acid molecule includes a nucleotide sequence described in SEQ ID NOs: 25, 26, 27, 28, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or a nucleotide sequence that is at least 90% identical thereto (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the aforementioned sequences).

[0242] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain and light chain variable regions of an antibody, the heavy chain and light chain variable regions comprising the amino acid sequence shown in SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20, SEQ ID NOs. 77 and 78, SEQ ID NOs. 79 and 80, SEQ ID NOs. 81 and 82, SEQ ID NOs. 83 and 84, SEQ ID NOs. 85 and 86, or SEQ ID NOs. 87 and 88, respectively, or an amino acid sequence that is at least 90% identical thereto (for example, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the aforementioned sequence).

[0243] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated to a viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can be directed to the expression of a gene to which they are functionally ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Hereinafter, “plasmid” and “vector” may be used interchangeably, as plasmids are the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0244] As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such a term is intended to refer not only to a specific target cell but also to the offspring of such a cell. Such offspring may not be identical to the parent cell in practice, as certain modifications may occur in later generations due to mutation or environmental influences, but are included within the scope of the term “host cell” as used herein.

[0245] G. Combination therapy Any of the binding domains, antibodies, their antigen-binding fragments, bispecific constructs, and / or multispecific constructs described herein may be administered in combination with additional therapies, i.e., in combination with other agents. As used herein, the term “concurrently administered” includes any or all of the simultaneous, separate, or sequential administrations (including administration as part of a drug regimen) of any of the binding domains, antibodies, their antigen-binding fragments, bispecific constructs, or multispecific constructs described herein in combination with adjuvants and other agents. For example, combination therapy may include administering any of the binding domains, antibodies, their antigen-binding fragments, bispecific constructs, and / or multispecific constructs described herein together with anti-inflammatory agents, DMARDs (disease-modifying antirheumatic drugs), immunosuppressants, chemotherapeutic agents, radiotherapy, other antibodies, cytotoxics, and / or drugs, as well as at least one additional therapeutic agent such as adjuvants, immunostimulants, and / or immunosuppressants.

[0246] In the treatment of tumors, chemotherapeutic agents suitable for co-administration with the binding domains, antibodies, their antigen-binding fragments, bispecific constructs and / or multispecific constructs described herein include, for example, Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. Further drugs include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin and anthramycin (AMC)), as well as mitotic inhibitors (e.g., vincristine and vinblastine) and temozolomide.

[0247] For example, agents that eliminate or inhibit immunosuppressive activity mediated by inhibitory factors produced by tumor or host cells within immune cells (e.g., regulatory T cells, NKT cells, macrophages, myeloid-derived suppressor cells, immature or suppressor dendritic cells) or the local microenvironment of a tumor (e.g., TGFβ, indoleamine 2,3-dioxygenase-IDO) may also be administered together with the binding domains, antibodies, their antigen-binding fragments, bispecific constructs and / or multispecific constructs described herein. Such agents include antibodies and small molecule drugs such as IDO inhibitors, such as 1-methyltryptophan or its derivatives.

[0248] Suitable agents for co-administration with the binding domains, antibodies, their antigen-binding fragments, bispecific constructs and / or multispecific constructs described herein for the treatment of such immune disorders include, for example, immunosuppressants such as rapamycin, cyclosporine and FK506, anti-TNF agents such as etanercept, adalimumab and infliximab, and steroids. Examples of certain natural and synthetic steroids include, for example, aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortibazole, deoxycorton, desonide, desoxymethasone, dexamethasone, fluoroocron, difluorocortolone, fluchlorolone, flumethasone, flunisolide, fluocinolone, fluocinolone, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flulandrenolone, fluticasone, halcinonide, hydrocortisone, icometasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, thixocortol, and triamcinolone.

[0249] Suitable agents for co-administration with the binding domains, antibodies, their antigen-binding fragments, bispecific constructs and / or multispecific constructs described herein for inducing or enhancing an immune response include, for example, adjuvants and / or immunostimulants, non-limiting examples of which have already been disclosed herein. In one embodiment, the immunostimulant is a TLR3 agonist such as polyIC.

[0250] As used herein, the term “immunostimulant” includes, but is not limited to, compounds that can stimulate dendritic cells (DCs) and antigen-presenting cells (APCs), such as macrophages. For example, immunostimulants suitable for use in the present invention can stimulate APCs, thereby accelerating the maturation process of APCs, increasing APC proliferation, and / or upregulating the recruitment or release of co-stimulating molecules (e.g., CD80, CD86, ICAM-1, MHC molecules, and CCR7) as well as pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-12, IL-15, and IFN-γ). Suitable immunostimulants can also increase T cell proliferation. Such immunostimulants include, but are not limited to, CD40 ligand, FLT3 ligand, cytokines such as IFN-α, IFN-β, IFN-γ, and IL-2, colony-stimulating factors such as G-CSF (granulocyte colony-stimulating factor) and GM-CSF (granulocyte-macrophage colony-stimulating factor), anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-41BB antibodies, or anti-OX-40 antibodies, LPS (endotoxin), ssRNA, dsRNA, Calmette-Guerin bacilli (BCG), levamisole hydrochloride, and intravenous immunoglobulins. In one embodiment, the immunostimulant may be a Toll-like receptor (TLR) agonist.For example, immunostimulants may be TLR3 agonists such as double-stranded inosine:cytosine polynucleotides (e.g., poly-I:C, available as Ampligen™ from Hemispherx Bipharma, PA, US) or poly-A:U; TLR4 agonists such as monophosphoryl lipid A (MPL) or RC-529 (e.g., available from GSK, UK); TLR5 agonists such as flagelline; imiquimod TLR7 or TLR8 agonists, such as imiquimod (e.g., Aldara™) or reximod and related imiquimod preparations (e.g., available from 3M Corporation); or TLR9 agonists such as deoxynucleotides having an unmethylated CpG motif (so-called "CpG", available from Coley Pharmaceuticals, for example). Such immunostimulants may be administered simultaneously, separately, or sequentially with the binding domains, antibodies, their antigen-binding fragments, bispecific constructs, and / or multispecific constructs described herein.

[0251] H. Use and Method of the Invention Methods for stimulating T cell activity, for inducing or enhancing an immune response, and for doing so by administering the bispecific constructs, multispecific constructs, antibodies, or antigen-binding fragments thereof described herein to patients who require treatment for a disease or condition (e.g., cancer).

[0252] As used herein, the terms “T cell-mediated response” or “T cell activity” refer to effector T cells (e.g., CD8). + (Cells) and helper T cells (e.g., CD4 +This refers to any response mediated by T cells, including T cells. T cell-mediated responses include, for example, T cell cytotoxicity and proliferation. Stimulation of T cell activity can be assessed using any of several indicators of T cell activity known in the art. For example, enhancement of interferon-gamma production by OKT3-stimulated T cells can be used as a measure of T cell activation. Stimulation of T cell activity can also be assessed using an NFκB-driven reporter gene system in CD27-expressing cells. Other suitable assays for T cell activation are well established in the art.

[0253] The terms "to induce an immune response" and "to enhance an immune response" are used interchangeably and refer to the stimulation of an immune response to a particular antigen (i.e., either passive or adaptive).

[0254] As used herein, the terms “to treat,” “to treat,” and “treatment” refer to the therapeutic or prophylactic measures described herein. A “treatment” method involves administering such treatments, bispecific constructs, multispecific constructs, antibodies, their antigen-binding fragments, or compositions described herein, to a subject requiring such treatment, for example, a subject requiring an enhanced immune response to a particular antigen, or a subject that may eventually acquire such a disorder, to prevent, cure, delay, reduce the severity thereof, or induce remission of one or more symptoms of a disorder or recurrent disorder, or to extend the survival of a subject beyond the expected survival in the absence of such treatment.

[0255] The term "effective dose" or "effective dosage" is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect. The term "therapeutic effective dose" is defined as the amount sufficient to cure, or at least partially prevent, the disease and its complications in a patient who already has the disease. The amount effective for this use will depend on the severity of the disorder being treated and the general state of the patient's own immune system.

[0256] The term “patient” includes human and other mammalian subjects receiving either preventive or therapeutic treatment.

[0257] As used herein, the term “inhibit growth” (for example, referring to cells) is intended to include any measurable reduction of cell growth, e.g., inhibition of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of cell growth.

[0258] In one embodiment, a method is provided for stimulating T cell activity, comprising contacting T cells with an antibody, or one of its antigen-binding fragments, bispecific constructs, multispecific constructs, or compositions described herein. Stimulating T cell activity may include, for example, stimulating IFN-gamma production.

[0259] In another embodiment, a method for inducing or enhancing an immune response (e.g., to an antigen) in a subject comprises administering to the subject an antibody, or one of its antigen-binding fragments, bispecific constructs, multispecific constructs, or compositions described herein, in an amount effective to induce or enhance an immune response (e.g., to an antigen) in the subject.

[0260] In another embodiment, a method is provided for treating a condition or disease in a subject, the method comprising administering to the subject an antibody, or one thereof, an antigen-binding fragment, a bispecific construct, a multispecific construct, or a composition described herein, in an amount effective for treating the condition or disease.

[0261] In another embodiment, a method is provided for treating a condition or disease in a subject, the method comprising administering to the subject one of the anti-CD27 antibodies or their antigen-binding fragments described herein in combination with one of the anti-PD-L1 antibodies or their antigen-binding fragments described herein. For example, in one embodiment, (i) The anti-CD27 antibody or its antigen-binding fragment is selected from the group consisting of (a) an anti-CD27 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 1, 2, and 3, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 4, 5, and 6, respectively, and (b) an anti-CD27 antibody or its antigen-binding fragment comprising the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 7, 8, and 9, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 10, 11, and 12, respectively. (ii) The anti-PD-L1 antibody or its antigen-binding fragment comprises (a) the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 29, 30, and 31, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 32, 33, and 34, respectively; (b) the anti-PD-L1 antibody or its antigen-binding fragment comprises the heavy chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 35, 36, and 37, respectively, and the light chain variable regions CDR1, CDR2, and CDR3 described in SEQ ID NOs. 38, 39, and 40, respectively; (c) the anti-PD-L1 antibody or its antigen-binding fragment comprises th...

Claims

1. It is an anti-CD27 antibody, (a) comprising heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 4, 5, and 6, respectively, or (b) comprising heavy chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 7, 8, and 9, respectively, and light chain variable regions CDR1, CDR2, and CDR3 described in Sequence IDs 10, 11, and 12, respectively Anti-CD27 antibody.

2. The antibody according to claim 1, (a) A heavy chain variable region including SEQ ID NO: 17 and a light chain variable region including SEQ ID NO: 18, or (b) A heavy chain variable region including SEQ ID NO: 19 and a light chain variable region including SEQ ID NO: 20, antibody.

3. The antibody according to claim 1 or 2, wherein the antibody is a human antibody.

4. The antibody according to any one of claims 1 to 3, wherein the antibody comprises a human IgG2 constant region.

5. A composition comprising the antibody and carrier according to any one of claims 1 to 4.

6. The composition according to claim 5, further comprising an adjuvant.

7. An isolated nucleic acid molecule comprising nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the antibody described in claim 1.

8. A vector comprising the nucleic acid molecule described in claim 7.

9. A host cell comprising the vector according to claim 8.

10. An in vitro method for stimulating T cell activity, comprising contacting T cells with an antibody according to any one of claims 1 to 4, or a composition according to claim 5 or 6.

11. A pharmaceutical composition for treating a condition or disease in a subject, comprising the antibody described in any one of claims 1 to 4.

12. The pharmaceutical composition according to claim 11, used in combination with an anti-PD-L1 antibody.

13. The pharmaceutical composition according to claim 12, wherein the composition and the anti-PD-L1 antibody are administered separately or simultaneously.

14. The pharmaceutical composition according to any one of claims 11 to 13, wherein the aforementioned condition or disease is cancer.

15. The pharmaceutical composition according to claim 14, wherein the cancer is colorectal cancer, ovarian cancer, renal cell carcinoma, head and neck squamous cell carcinoma, or glioblastoma.

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