Single-domain antibodies and variants thereof against CTLA-4

1. An isolated anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) construct comprising a single-domain antibody (sdAb) moiety specifically recognizing CTLA-4 (anti-CTLA-4 sdAb moiety), wherein the anti-CTLA-4 sdAb moiety comprises any one of the following:

(1) a CDR1 comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 88;

(2) a CDR1 comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 82;

(3) a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 90;

(4) a CDR1 comprising the amino acid sequence of SEQ ID NO: 213, a CDR2 comprising the amino acid sequence of SEQ ID NO: 233, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 253;

(5) a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 49, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 81;

(6) a CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 83;

(7) a CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 84;

(8) a CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 85;

(9) a CDR1 comprising the amino acid sequence of SEQ ID NO: 22, a CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 86;

(10) a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 87;

(11) a CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR2 comprising the amino acid sequence of SEQ ID NO: 57, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 89;

(12) a CDR1 comprising the amino acid sequence of SEQ ID NO: 27, a CDR2 comprising the amino acid sequence of SEQ ID NO: 59, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 91;

(13) a CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 comprising the amino acid sequence of SEQ ID NO: 60, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 92;

(14) a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 93;

(15) a CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 94;

(16) a CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR2 comprising the amino acid sequence of SEQ ID NO: 63, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 95;

(17) a CDR1 comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 comprising the amino acid sequence of SEQ ID NO: 64, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 96;

(18) a CDR1 comprising the amino acid sequence of SEQ ID NO: 214, a CDR2 comprising the amino acid sequence of SEQ ID NO: 234, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 254;

(19) a CDR1 comprising the amino acid sequence of SEQ ID NO: 215, a CDR2 comprising the amino acid sequence of SEQ ID NO: 235, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 255;

(20) a CDR1 comprising the amino acid sequence of SEQ ID NO: 216, a CDR2 comprising the amino acid sequence of SEQ ID NO: 236, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 256;

(21) a CDR1 comprising the amino acid sequence of SEQ ID NO: 217, a CDR2 comprising the amino acid sequence of SEQ ID NO: 237, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 257;

(22) a CDR1 comprising the amino acid sequence of SEQ ID NO: 218, a CDR2 comprising the amino acid sequence of SEQ ID NO: 238, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 258;

(23) a CDR1 comprising the amino acid sequence of SEQ ID NO: 219, a CDR2 comprising the amino acid sequence of SEQ ID NO: 239, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 259;

(24) a CDR1 comprising the amino acid sequence of SEQ ID NO: 220, a CDR2 comprising the amino acid sequence of SEQ ID NO: 240, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 260;

(25) a CDR1 comprising the amino acid sequence of SEQ ID NO: 221, a CDR2 comprising the amino acid sequence of SEQ ID NO: 241, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 261;

(26) a CDR1 comprising the amino acid sequence of SEQ ID NO: 222, a CDR2 comprising the amino acid sequence of SEQ ID NO: 242, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 262; and

(27) a CDR1 comprising the amino acid sequence of SEQ ID NO: 214, a CDR2 comprising the amino acid sequence of SEQ ID NO: 339, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 254.

2. The isolated anti-CTLA-4 construct of claim 1 , wherein the anti-CTLA-4 sdAb moiety comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 113-129, 200-202, 274-282, 341-344, 352, and 353, or a variant thereof having at least 90% sequence identity to any one of SEQ ID NOs: 113-129, 200-202, 274-282, 341-344, 352, and 353.

3. The isolated anti-CTLA-4 construct of claim 1 , wherein the anti-CTLA-4 sdAb moiety is camelid, chimeric, partially humanized, or fully humanized.

4. The isolated anti-CTLA-4 construct of claim 1 , wherein the isolated anti-CTLA-4 construct is a heavy chain-only antibody (HCAb).

5. The isolated anti-CTLA-4 construct of claim 4 , wherein the HCAb comprises the amino acid sequence of any one of SEQ ID NOs: 130-133, 283-291, and 366-371.

6. The isolated anti-CTLA-4 construct of claim 1 , wherein the isolated anti-CTLA-4 construct further comprises a second antibody moiety specifically recognizing a second epitope.

7. The isolated anti-CTLA-4 construct of claim 6 , wherein the second antibody moiety is a full-length antibody, a Fab, a Fab′, a (Fab′)2, an Fv, a single chain Fv (scFv), an scFv-scFv, a minibody, a diabody, or an sdAb.

8. The isolated anti-CTLA-4 construct of claim 6 , wherein the anti-CTLA-4 sdAb moiety and the second antibody moiety are connected by a peptide linker.

9. The isolated anti-CTLA-4 construct of claim 8 , wherein the peptide linker comprises the amino acid sequence of SEQ ID NO: 162, 163, 307, or 365.

10. The isolated anti-CTLA-4 construct of claim 7 , wherein the second antibody moiety is a full-length antibody consisting of two heavy chains and two light chains.

11. The isolated anti-CTLA-4 construct of claim 10 , comprising a configuration selected from the group consisting of:

(1) the N-terminus of the anti-CTLA-4 sdAb moiety is fused to the C-terminus of at least one of the heavy chains of the full-length antibody;

(2) the C-terminus of the anti-CTLA-4 sdAb moiety is fused to the N-terminus of at least one of the heavy chains of the full-length antibody;

(3) the N-terminus of the anti-CTLA-4 sdAb moiety is fused to the C-terminus of at least one of the light chains of the full-length antibody;

(4) the C-terminus of the anti-CTLA-4 sdAb moiety is fused to the N-terminus of at least one of the light chains of the full-length antibody;

(5) the isolated anti-CTLA-4 construct comprises four anti-CTLA-4 sdAb moieties, wherein the C-terminus of each anti-CTLA-4 sdAb moiety is fused to the N-terminus of each chain of the full-length antibody; and

(6) the isolated anti-CTLA-4 construct comprises four anti-CTLA-4 sdAb moieties, wherein the C-terminus of the first anti-CTLA-4 sdAb moiety is fused to the N-terminus of the second anti-CTLA-4 sdAb moiety, wherein the C-terminus of the second anti-CTLA-4 sdAb moiety is fused to the N-terminus of the first heavy chain of the full-length antibody, wherein the C-terminus of the third anti-CTLA-4 sdAb moiety is fused to the N-terminus of the fourth anti-CTLA-4 sdAb moiety, and wherein the C-terminus of the fourth anti-CTLA-4 sdAb moiety is fused to the N-terminus of the second heavy chain of the full-length antibody.

12. The isolated anti-CTLA-4 construct of claim 10 , wherein the full-length antibody specifically recognizes programmed cell death protein 1 (PD-1) (anti-PD-1 full-length antibody), and wherein the anti-PD-1 full-length antibody comprises heavy chain complementarity-determining regions (HC-CDRs) and light chain complementarity-determining regions (LC-CDRs) of:

(1) a heavy chain comprising the amino acid sequence of SEQ ID NO: 158, and a light chain comprising the amino acid sequence of SEQ ID NO: 159;

(2) a heavy chain comprising the amino acid sequence of SEQ ID NO: 160, and a light chain comprising the amino acid sequence of SEQ ID NO: 161; or

(3) a heavy chain comprising the amino acid sequence of SEQ ID NO: 308, and a light chain comprising the amino acid sequence of SEQ ID NO: 309.

13. The isolated anti-CTLA-4 construct of claim 10 , wherein the full-length antibody specifically recognizes programmed death-ligand 1 (PD-L1) (anti-PD-L1 full-length antibody), and wherein the anti-PD-L1 full-length antibody comprises HC-CDRs and LC-CDRs of:

(1) a heavy chain comprising the amino acid sequence of SEQ ID NO: 195, and a light chain comprising the amino acid sequence of SEQ ID NO: 196; or

(2) a heavy chain comprising the amino acid sequence of SEQ ID NO: 197, and a light chain comprising the amino acid sequence of SEQ ID NO: 198.

14. The isolated anti-CTLA-4 construct of claim 11 , wherein the full-length antibody specifically recognizes PD-1 (anti-PD-1 full-length antibody),

(i) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159; and wherein the anti-CTLA-4 sdAb moiety comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 114, 129, 274, 275, 278, 280, and 282;

(ii) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid of SEQ ID NO: 160, and two light chains each comprising the amino acid sequence of SEQ ID NO: 161; and wherein the anti-CTLA-4 sdAb moiety comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 114, 129, 274, 275, 278, 280, and 282; or

(iii) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 308, and two light chains each comprising the amino acid sequence of SEQ ID NO: 309; and wherein the anti-CTLA-4 sdAb moiety comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 274, 275, 278, 280, 282, and 341-344.

15. The isolated anti-CTLA-4 construct of claim 11 , wherein the full-length antibody specifically recognizes PD-L1 (anti-PD-L1 full-length antibody),

(i) wherein the anti-PD-L1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 195, and two light chains each comprising the amino acid sequence of SEQ ID NO: 196; and wherein the anti-CTLA-4 sdAb moiety comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 114, 129, 274, 275, 278, 280, and 282; or

(ii) wherein the anti-PD-L1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 197, and two light chains each comprising the amino acid sequence of SEQ ID NO: 198; and wherein the anti-CTLA-4 sdAb moiety comprises a V H H domain comprising the amino acid sequence of SEQ ID NO: 114 or 129.

16. The isolated anti-CTLA-4 construct of claim 14 ,

(i) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159, wherein at least one of the heavy chains of the anti-PD-1 full-length antibody is fused to the anti-CTLA-4 sdAb moiety, and wherein the heavy chain fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 134-145, 292-296, and 319-323;

(ii) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 160, and two light chains each comprising the amino acid sequence of SEQ ID NO: 161, wherein at least one of the heavy chains of the anti-PD-1 full-length antibody is fused to the anti-CTLA-4 sdAb moiety, and wherein the heavy chain fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 146-157, 297-301, and 324-328;

(iii) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 308, and two light chains each comprising the amino acid sequence of SEQ ID NO: 309, wherein at least one of the heavy chains of the anti-PD-1 full-length antibody is fused to the anti-CTLA-4 sdAb moiety, and wherein the heavy chain fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 310-318 and 329-337;

(iv) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159, wherein at least one of the light chains of the anti-PD-1 full-length antibody is fused to the anti-CTLA-4 sdAb moiety, and wherein the light chain fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 354 or 355;

(v) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159, wherein the isolated anti-CTLA-4 construct comprises four anti-CTLA-4 sdAb moieties, wherein the C-terminus of each anti-CTLA-4 sdAb moiety is fused to the N-terminus of each chain of the anti-PD-1 full-length antibody, and wherein the heavy chain fusion polypeptides each comprises the amino acid sequence of SEQ ID NO: 356, and the light chain fusion polypeptides each comprises the amino acid sequence of SEQ ID NO: 357; or

(vi) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159, wherein the isolated anti-CTLA-4 construct comprises four anti-CTLA-4 sdAb moieties, wherein the C-terminus of the first anti-CTLA-4 sdAb moiety is fused to the N-terminus of the second anti-CTLA-4 sdAb moiety, wherein the C-terminus of the second anti-CTLA-4 sdAb moiety is fused to the N-terminus of the first heavy chain of the anti-PD-1 full-length antibody, wherein the C-terminus of the third anti-CTLA-4 sdAb moiety is fused to the N-terminus of the fourth anti-CTLA-4 sdAb moiety, and wherein the C-terminus of the fourth anti-CTLA-4 sdAb moiety is fused to the N-terminus of the second heavy chain of the anti-PD-1 full-length antibody, and wherein the heavy chain fusion polypeptides each comprises the amino acid sequence of SEQ ID NO: 358.

17. The isolated anti-CTLA-4 construct of claim 15 ,

(i) wherein the anti-PD-L1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 195, and two light chains each comprising the amino acid sequence of SEQ ID NO: 196, wherein at least one of the heavy chains of the anti-PD-L1 full-length antibody is fused to the anti-CTLA-4 sdAb moiety, and wherein the heavy chain fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 171-182, 302-306, and 345-349; or

(ii) wherein the anti-PD-L1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 197, and two light chains each comprising the amino acid sequence of SEQ ID NO: 198, wherein at least one of the heavy chains of the anti-PD-L1 full-length antibody is fused to the anti-CTLA-4 sdAb moiety, and wherein the heavy chain fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 183-194.

18. The isolated anti-CTLA-4 construct of claim 14 , wherein each heavy chain of the anti-PD-1 full-length antibody is fused to an anti-CTLA-4 sdAb moiety,

(i) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159, and wherein each heavy chain fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 292;

(ii) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159, and wherein each heavy chain fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 293;

(iii) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 158, and two light chains each comprising the amino acid sequence of SEQ ID NO: 159, and wherein each heavy chain fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 294;

(iv) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 160, and two light chains each comprising the amino acid sequence of SEQ ID NO: 161, and wherein each heavy chain fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 297;

(v) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 160, and two light chains each comprising the amino acid sequence of SEQ ID NO: 161, and wherein each heavy chain fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 298; or

(vi) wherein the anti-PD-1 full-length antibody comprises two heavy chains each comprising the amino acid sequence of SEQ ID NO: 160, and two light chains each comprising the amino acid sequence of SEQ ID NO: 161, and wherein each heavy chain fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 299.

19. The isolated anti-CTLA-4 construct of claim 1 , wherein the anti-CTLA-4 sdAb moiety comprises a V H H domain comprising the amino acid sequence of any one of SEQ ID NOs: 123, 274, 275, and 280.

20. A method of treating an individual having a CTLA-4 positive cancer, comprising administering to the individual an effective amount of the isolated anti-CTLA-4 construct of claim 3 .

21. The method of claim 20 , wherein the CTLA-4 positive cancer is colon cancer.