Anti-TIGIT antibodies and methods of use

Abstract

The present invention relates to anti-TIGIT antibodies and methods of use thereof, including multispecific anti-TIGIT antibodies that bind to the "Ig and ITIM domain" T cell immunoreceptor (TIGIT) and have binding specificity for TIGIT and one or more additional antigens. In some embodiments, these anti-TIGIT antibodies include single domain antibodies that bind to TIGIT.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Chinese Patent Application No. CN202010024565.9, filed on January 10, 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to antibodies that bind to a T cell immunoreceptor (TIGIT) having Ig and ITIM domains, including multispecific anti-TIGIT antibodies that have binding specificity for TIGIT and one or more additional antigens, and methods for using the same. [Background technology]

[0003] The T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an immune checkpoint receptor expressed by immune cells (e.g., activated T cells and natural killer cells (NK cells)) and mediates immune inhibition. The ligand for TIGIT contains PVR (CD155), which has been identified on dendritic cells (DCs), macrophages, and many human cancer cells. It has been shown to downregulate T cell activation and cytokine secretion after binding to TIGIT. Inhibition of TIGIT / PVR interaction can mediate effective antitumor activity of immune cells. Given the important role of TIGIT in immune checkpoint regulation, there is still a need in the art to develop therapeutic molecules and methods for immunotherapy and cancer treatment that modulate immune cells mediated by TIGIT. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides isolated monoclonal antibodies that specifically bind to TIGIT with high affinity, including multispecific antibodies that bind to TIGIT and one or more additional targets. In some embodiments, the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT. The disclosure further provides methods for preparing the antibodies, immunoconjugates, and pharmaceutical compositions comprising these antibodies, and methods for using the antibodies, immunoconjugates, and pharmaceutical compositions comprising these antibodies to treat, for example, diseases and conditions (e.g., cancer). The present invention is based, in part, on the discovery of single domain anti-TIGIT antibodies that bind to TIGIT, which can increase immune responses in immune cells and provide improved anti-tumor effects.

[0005] In some embodiments, the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT. In some embodiments, the single domain antibody is present in an amount of about 1×10 -7 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 1×10 -8 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 5×10 -9 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 2×10 -9 Binds to TIGIT with a KD of M or smaller.

[0006] In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 96. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 98, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 99, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 100. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 102, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 103, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 104. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 106, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 107, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 108. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 110, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 111, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 112.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 114, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 115, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 116. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 118, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 119, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 120. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 122, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 123, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 124. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 126, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 127, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 128. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 130, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 136. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 138, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 139, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 140. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 142, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 143, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 146, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 147, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 148. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 150, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 151, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 152.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 154, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 156. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 158, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 159, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 160. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 162, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 163, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 164. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 166, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 168. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 170, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 171, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 172.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 174, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 175, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 176. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 178, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 182, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 183, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 184. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 186, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 187, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 188. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 190, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192.

[0007] In some embodiments, the single domain antibody comprises a heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, and 190, or a variant thereof having up to about three amino acid substitutions; and b) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, and 190, or a variant thereof having up to about three amino acid substitutions. and c) a heavy chain variable region CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, and 192, or a variant thereof having up to about three amino acid substitutions.

[0008] In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain, and the CDR3 domain respectively comprise the CDR1 domain, the CDR2 domain, and the CDR3 domain comprise the CDR1 domain, the CDR2 domain, and the CDR3 domain comprise the CDR3 domain contained in a reference heavy chain variable region, and the reference heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, and 193.

[0009] In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 98, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 99, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 102, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 103, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 104. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 106, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 107, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 108. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 110, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 111, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 112. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 114, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 115, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 116. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 118, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 119, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 120.In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 122, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 123, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 124. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 126, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 127, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 128. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 130, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 136. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 138, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 139, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 140. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 142, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 143, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 146, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 147, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 148.In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 150, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 151, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 152. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 154, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 156. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 158, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 159, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 160. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 162, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 163, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 164. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 166, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 168. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 170, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 171, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 172. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 174, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 175, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 176.In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 178, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 182, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 183, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 184. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 186, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 187, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 188. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 190, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192.

[0010] In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, and 193.

[0011] In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 105. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 117. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 133. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 141. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 145. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 149. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 153. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 157.In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 161. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 165. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 169. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 177. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 181. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 185. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 189. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 193.

[0012] In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 5, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 6, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 7. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 11. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 13, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 14, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 17, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 18, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 19.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 23. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 25, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 26, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 27. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 29, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 30, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 31. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 33, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 34, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 35. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 37, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 38, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 39.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 45, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 46, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 47. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 49, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 50, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 51. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 53, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 54, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 55. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 57, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 58, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 59.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 63. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 65, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 66, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 67. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 69, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 70, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 71. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 74, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 75. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 77, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 78, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 79.In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 81, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 82, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 83.

[0013] In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 5, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 6, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 7. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 11. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 13, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 14, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 17, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 18, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 19. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 23.In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 25, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 26, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 27. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 29, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 30, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 31. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 33, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 34, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 35. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 37, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 38, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 39. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 45, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 46, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 47.In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 49, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 50, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 51. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 53, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 54, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 55. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 57, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 58, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 59. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 63. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 65, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 66, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 67. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 69, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 70, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 71.In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 74, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 75. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 77, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 78, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 79. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 81, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 82, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 83.

[0014] In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, and 84. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, and 84.

[0015] In some embodiments, the single domain antibody comprises a humanized framework.

[0016] In some embodiments, the anti-TIGIT antibody comprises an Fc region. In some embodiments, the Fc region comprises a human Fc region. In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In some embodiments, the Fc region comprises an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, Y300L, and P396L. In some embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E. In some embodiments, the anti-TIGIT antibody comprises the amino acid sequence set forth in SEQ ID NO:194.

[0017] In some embodiments, the heavy chain variable region is linked to the Fc region via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises about 4 to about 30 amino acids. In some embodiments, the peptide linker comprises about 4 to about 15 amino acids. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220.

[0018] In some embodiments, the anti-TIGIT antibody comprises a multispecific antibody, e.g., a bispecific antibody, a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[0019] In some embodiments, the anti-TIGIT antibody comprises a multispecific antibody (e.g., a bispecific antibody), which comprises a second antibody portion that specifically binds to a second antigen. In some embodiments, the second antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from the group consisting of Her-2, EGFR, PD-L1, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein ( EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA 1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), inclusion protein 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), tyrosine kinase transmembrane receptor 1 (ROR1), and any combination thereof.

[0020] In some embodiments, the second antigen is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is selected from the group consisting of PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof.

[0021] In some embodiments, the anti-TIGIT antibody is conjugated to a therapeutic agent or label. In some embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0022] The present disclosure further provides an immunoconjugate comprising any of the antibodies disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin. In some embodiments, the therapeutic agent is a radioisotope.

[0023] The present disclosure further provides pharmaceutical compositions, in some embodiments, comprising: a) an antibody or immunoconjugate disclosed herein; and b) a pharmaceutically acceptable carrier agent.

[0024] The present disclosure further provides nucleic acids encoding any of the antibodies disclosed herein. The present disclosure further provides vectors comprising any of the nucleic acids disclosed herein. The present disclosure further provides host cells comprising a nucleic acid or vector disclosed herein.

[0025] The present disclosure further provides methods for preparing the antibodies disclosed herein, in some embodiments, the methods comprising expressing the antibody in a host cell disclosed herein and isolating the antibody from the host cell.

[0026] The present disclosure further provides methods for reducing tumor burden in a subject, in some embodiments, comprising administering to the subject an effective amount of an antibody, immunoconjugate, or drug composition disclosed herein.

[0027] In some embodiments, the method reduces the number of tumor cells. In some embodiments, the method reduces tumor size. In some embodiments, the method eradicates the tumor in the subject. In some embodiments, the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

[0028] The present disclosure further provides methods for treating and / or preventing a neoplasm in a subject, in some embodiments, the methods comprising administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0029] The present disclosure further provides methods for extending survival of a subject having a neoplasm, in some embodiments, the methods comprising administering to the subject an effective amount of an antibody, immunoconjugate, or pharmaceutical composition disclosed herein.

[0030] In some embodiments, the neoplasm is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0031] The present disclosure further provides any antibody disclosed herein for use as a drug. The present disclosure further provides any antibody disclosed herein for use in treating cancer. The present disclosure further provides a drug composition disclosed herein for use as a drug. The present disclosure further provides a drug composition disclosed herein for use in treating cancer. In some embodiments, the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

[0032] The present disclosure further provides kits comprising the antibodies, immunoconjugates, pharmaceutical compositions, nucleic acids, vectors, or host cells disclosed herein. In some embodiments, the kits include instructions for treating and / or preventing neoplasia. [Brief explanation of the drawings]

[0033] [Figure 1A] The whole-cell binding of representative VHH bivalent antibodies to human TIGIT as determined by flow cytometry assay is depicted. Figure 1A shows measurements relative to a reference anti-human TIGIT antibody (Reference Ab 1). Figure 1B shows different binding profiles using other VHH antibodies. The Y-axis represents the mean fluorescence intensity of AlexaFlour 488. The X-axis represents the antibody concentration in nanomolar units. 2B7, 1G1, 1C12, 3G6, 2B10, 3G7, 3G10, 13H11, and 15A5 are anti-human TIGIT VHH clones. EC50 values ​​were obtained using the nonlinear regression method in Prism, and these values ​​are shown in nanomolar in the table. Figure 1C is a structural schematic of the TIGIT VHH bivalent antibodies. [Figure 1B] Same as above. [Figure 1C] Same as above. [Figure 2A] The effectiveness of representative bivalent antibodies in blocking TIGIT activity is plotted, as determined by luciferase reporter gene assay. Jurkat cells stably transfected with human TIGIT and an NFAT reporter gene are co-cultured with Raji cells stably transfected with PVR (CD155) in the presence of a representative anti-TIGIT bivalent antibody and low concentrations of staphylococcal enterotoxin. The Y-axis represents NFAT luciferase activity in relative luminescence units. The X-axis represents antibody concentration in nanomoles. Reference Ab 1 is a reference anti-h-TIGIT antibody. 2B7, 1G1, 1C12, 3G6, 2B10, 3G7, and 3F10 are representative anti-h-TIGIT clones. EC50 values ​​were obtained using the nonlinear regression method in Prism, and these values ​​are shown in nanomoles in the table. [Figure 2B] Same as above. [Figure 3A]The whole cell binding of the humanized 1C12 and 1G1 clones to human TIGIT as determined by flow cytometry assay is plotted. Representative results for the 1C12 humanized format are shown in Figure 3A. The 1C12 chimeric antibody is an antibody containing the Llama VHH sequence of the 1C12 clone and the CH2 and CH3 domains of human IgG1. 1C12(F-EREF), 1C12(F-EREW), and 1C12(F-GLEW) are humanized versions of the 1C12 clone, differing by mutations in framework 2. Representative results for the 1G1 humanized format are shown in Figure 3B. The 1G1 chimeric antibody is an antibody containing the Llama VHH sequence of the 1G1 clone and the CH2 and CH3 domains of human IgG1. 1G1(FG-ERES), 1G1(FA-ERES), 1G1(FA-EREW), and 1G1(FA-GLEW) are four different forms of the humanized 1G1 clone with different mutations in framework 2. The Y-axis is the mean fluorescence intensity value of AlexaFlour 488. The X-axis is the antibody concentration value in nanomolar units. [Figure 3B] Same as above. [Figure 4A] The effectiveness of humanized 1C12 and 1G1 clones in blocking TIGIT activity is described, as determined by luciferase reporter gene assay. Representative results for the 1C12 humanized format in the TIGIT-blocking luciferase reporter gene assay are shown in Figure 4A. Compared to the reference anti-h-TIGIT antibody, reference Ab 1, all clones are more effective. Representative results for the 1G1 humanized format are shown in Figure 4B. The Y-axis represents NFAT luciferase activity (RLU, relative luminescence units). The X-axis represents antibody concentration in nanomolar units. [Figure 4B] Same as above. [Figure 5] The correlation between IC50 values ​​(in nanomolar) from blocking ELISA and EC50 values ​​(in nanomolar) from whole cell binding for representative clones is plotted. Clone names are marked in the accompanying figures. The correlation was analyzed using GraphPad Prism. [Figure 6A]The thermal stability of representative clones tested is plotted. VHH antibody samples were heated from 25°C to 70°C for 60 minutes. Binding of heated samples to human TIGIT was examined using ELISA or whole cell binding. Figure 6A shows the binding of heated samples of representative clones to h-TIGIT ECD in an ELISA assay, where clone names are marked in the accompanying drawings. The Y-axis represents the OD450 obtained by ELISA assay. The X-axis represents the treatment temperature. Figure 6B shows the binding of heated samples of representative clones to h-TIGIT stably expressed in Jurkat cells, as determined by flow cytometry. The Y-axis represents the percentage of binding to h-TIGIT. The X-axis represents the treatment temperature. [Figure 6B] Same as above. [Figure 7A] The binding epitope of 2A3-Fc to human TIGIT, determined by ForteBio's Octet binding assay, is depicted. Recombinant human TIGIT ECD (200 nM) protein with a His tag is loaded onto the sensor. Binding is detected by injecting three different concentrations of 2A3-Fc. While no additional binding is detected for 2A3-Fc after a second injection (Figure 7A), binding is detected after injecting three different concentrations of reference Ab 2, a single reference anti-TIGIT antibody (as shown in Figure 7B), or reference Ab 1 (as shown in Figure 7C). The results show that the 2A3 clone has a different binding epitope compared to reference Ab 2 and reference Ab 1. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8A]Figure 8 depicts the cross-binding activity of 2A3-Fc to human, cynomolgus monkey, and mouse TIGIT as determined by ELISA assay. Figure 8A shows the binding of 2A3-Fc to recombinant human TIGIT ECD. Reference Ab 2 is an anti-human TIGIT reference antibody. Both 2A3-Fc and reference Ab 2 bind to h-TIGIT with similar affinities. Anti-PDL1 does not bind to h-TIGIT. Figure 8B shows the binding of 2A3-Fc to recombinant cyno-TIGIT. Both 2A3-Fc and reference Ab 2 bind to cyno-TIGIT with similar affinities. Anti-PDL1 does not bind to cyno-TIGIT. Figure 8C shows the binding of 2A3-Fc to recombinant mouse TIGIT. Neither 2A3-Fc nor reference Ab 2 binds to mouse TIGIT, whereas an anti-mouse TIGIT reference antibody (Biolegend #142101) binds to mouse TIGIT with high affinity. The Y-axis represents OD450, and the X-axis represents antibody concentration in μg / ml. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9] Figure 1 depicts a comparison of the potency of representative clone 2A3-Fc and 2A3-LT-Fc in hot spot correction format in whole cell binding assays and human TIGIT blocking reporter gene assays. The Y axis represents mean fluorescence intensity in the attached figures above, and NFAT luciferase reporter gene activity in relative luminescence units in the attached figures below. The X axis represents antibody concentration in nanomolar units. 2A3-LT-Fc in hot spot correction format has similar potency to the parent clone 2A3-Fc. [Figure 10A]Figure 10A shows a comparison of the affinity of 2A3-LT-Fc and the reference anti-h-TIGIT antibody, Reference Ab 2, in a hot spot correction format. Figure 10A shows a comparison of the potency of 2A3-LT-Fc and Reference Ab 2 in a hot spot correction format in whole cell binding. The Y-axis represents the mean fluorescence intensity of AlexaFlour 488 as determined by flow cytometry assay using CytoFlex. The X-axis represents antibody concentration in nanomolar units. Data shown are representative results from three separate experiments. The affinity of 2A3-LT-Fc for h-TIGIT is slightly higher than that of Reference Ab 2. Figure 10B shows a comparison of the potency of 2A3-LT-Fc and Reference Ab 2 in a hot spot correction format in a TIGIT-blocking NFAT reporter gene assay. The Y-axis represents NFAT luciferase reporter gene activity in relative luminescence units. The X-axis represents antibody concentration in nanomolar units. 2A3-LT-Fc has similar potency to the reference Ab 2. [Figure 10B] Same as above. [Figure 11] To demonstrate the in vitro antitumor efficacy of 2A3-LT-Fc, TIGIT+ T cells and PVR+ dendritic cells (DCs) were used in a mixed lymphocyte reaction assay. After 48 hours of co-culture, T cell IL-2 secretion was detected in the culture supernatant. Anti-PD1 antibody was used as a positive control. Anti-HER2 antibody was used as a negative control. [Figure 12]Figure 1 depicts the binding of 2A3-LT-Fc wild-type (wt) and Fc mutants (DLE and VLPLL) to human FcγRIIIA, human FcγRIIB, and mouse FcγRIV as determined by Octet binding assay. The sensor was loaded with recombinant ECD proteins of FcγRIIIA, human FcγRIIB, and mouse FcγRIV, and association and dissociation of five different concentrations of 2A3-LT-Fc wt, DLE, or VLPLL mutants was detected using ForteBio. The Y-axis represents association, dissociation, and Rmax. The X-axis represents time in seconds. Compared to the wild-type, both the DLE and VLPLL mutants had enhanced binding affinity to human FcγRIIIA, and the DLE mutant also had enhanced binding affinity to human FcγRIIB, while the VLPLL mutant had reduced binding affinity to human FcγRIIB. All formats had similar affinities to mouse FcγRIV. [Figure 13A]Figure 13A depicts the effect of TIGIT mAb 2A3-LT-Fc wt and DLE mutants on blockade of TIGIT activity and human FcγRIIIA-mediated activity, respectively. Figure 13B depicts the effect of TIGIT mAb 2A3-LT-Fc wt and DLE mutants on blockade of TIGIT activity, as determined using an NFAT luciferase reporter gene assay. Jurkat cells stably transfected with human TIGIT and an NFAT reporter gene were co-cultured with Raji cells stably transfected with PVR. 2A3-LT-Fc wt and mutants were added and cultured for 5 hours. TCR-mediated activity was measured by luciferase activity. The Y-axis represents NFAT luciferase activity in relative luminescence units. The X-axis represents antibody concentration in nanomolar units. Figure 13B depicts the effect of TIGIT mAb 2A3-LT-Fc wt, DLE mutant on human FcγRIIIA-mediated activity, as determined by FcγRIIIA-mediated NFAT luciferase reporter gene activity. Jurkat cells stably transfected with human FcγRIIIA and NFAT were co-cultured with 293T cells stably transfected with human TIGIT for 5 hours in the presence of different concentrations of 2A3-LT-Fc wt, DLE mutant. Luciferase activity was measured and is represented in relative luminescence units on the Y-axis. The X-axis represents antibody concentration in nanomolar. [Figure 13B] Same as above. [Figure 14A]Figure 14 depicts the in vivo efficacy of anti-TIGIT antibodies. Human TIGIT knock-in C57BL / 6 mice and the MC38 mouse colon cancer model were used. Mice were inoculated with MC38 tumor cells one week before treatment. When the average tumor size reached approximately 51 mm, treatment began. Treatment was administered intraperitoneally twice weekly for 2.5 weeks. The 2A3-LT-Fc antibody was administered at 6 mg / kg per dose, or reference Ab 2 was administered at 11 mg / kg per dose (same mole / kg as the 2A3-LT-Fc antibody). Figure 14A depicts tumor growth curves for tumor-bearing mice treated with vehicle control, 2A3-Fc-wt, 2A3-Fc with the DLE mutation, and reference Ab 2. Figure 14B shows the results of single tumor volume. Figure 14C demonstrates that there was no significant change in body weight among the experimental groups in this study. [Figure 14B] Same as above. [Figure 14C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure provides isolated monoclonal antibodies that specifically bind to TIGIT with high affinity, including multispecific antibodies that bind to TIGIT and one or more additional targets. In some embodiments, the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT. The disclosure further provides methods for preparing the antibodies, immunoconjugates, and pharmaceutical compositions comprising these antibodies, and methods for using the antibodies, immunoconjugates, and pharmaceutical compositions comprising these antibodies to treat, for example, diseases and conditions (e.g., cancer). The present invention is based, in part, on the discovery of single domain anti-TIGIT antibodies that bind to TIGIT, which can increase immune responses in immune cells and provide improved anti-tumor effects.

[0035] For clarity, and not limitation, specific embodiments of the presently disclosed subject matter are divided as follows.

[0036] 1. Definition, 2. Antibodies, 3.How to use, 4. Drug formulations, and 5.Product.

[0037] 1.Definition As used herein, the term "antibody" is used in the broadest sense and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single domain antibodies, and antibody fragments, as long as they exhibit the required antigen-binding activity.

[0038] "Antibody fragment" refers to a molecule comprising an antigen-binding portion of an intact, full-length antibody, which binds to the same antigen as the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), multispecific antibodies formed from antibody fragments, single-domain antibodies, VHH nanobodies, domain antibodies, bivalent domain antibodies, or any other fragment of an antibody that binds to an antigen. "VHH" refers to a single-domain antibody isolated from a camelid. In some embodiments, the VHH comprises the heavy chain variable region of a camelid heavy chain antibody. In some embodiments, the size of the VHH does not exceed 25 kDa. In some embodiments, the size of the VHH does not exceed 20 kDa. In some embodiments, the size of the VHH does not exceed 15 kDa.

[0039] A "full-length antibody" refers to an antibody comprising two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the heavy and light chains may be referred to as "VH" and "VL," respectively. The variable regions in the two chains generally comprise three highly variable loops, which are called complementarity-determining regions (CDRs) (light chain (LC) CDRs comprising LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs comprising HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies and antigen-binding fragments disclosed herein may be defined or identified according to well-known conventions, such as those of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of a heavy or light chain are interposed between flanking segments called framework regions (FRs), which are more conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit multiple effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains. Several major antibody classes are divided into subclasses, for example, IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgG1 (α1 heavy chain), or IgG2 (α2 heavy chain).

[0040] An antibody that "cross-competes for binding" with a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay; conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0041] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. The fragment consists of a dimer of one heavy-chain variable region and one light-chain variable region in tight, non-covalent association. Folding of the two domains releases six hypervariable loops (three loops per heavy chain and three loops per light chain), which provide the amino acid residues for antigen binding and confer the antibody's binding specificity for the antigen. However, even a single variable domain (or half an Fv containing only three CDRs specific for an antigen) can recognize and bind to an antigen, albeit with lower affinity than the complete binding site.

[0042] "Single-chain Fv" (also abbreviated as "sFv" or "scFv") refers to VFvs linked to a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment comprising a V H and V L The scFv further comprises a polypeptide linker between the domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0043] For purposes of this specification, a "recipient human framework" or "human framework" is a framework that includes the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human shared framework. A recipient human framework that is "derived" from a human immunoglobulin framework or a human shared framework may include the same amino acid sequence or may include amino acid sequence changes. In some embodiments, the number of amino acid changes may be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL recipient framework and the VL human immunoglobulin framework sequence or the human shared framework sequence are identical in terms of sequence.

[0044] "Affinity" refers to the strength of the combined non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y may typically be expressed as a dissociation constant (KD). Affinity may be measured by conventional methods known in the art, including those described herein. Specific descriptions and illustrative examples for measuring binding affinity are provided below.

[0045] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more CDRs or hypervariable regions (HVRs), relative to a parent antibody that does not possess such modifications, which modifications provide the antibody with improved affinity for antigen.

[0046] As used herein, "T cell immunoreceptor having Ig and ITIM domains" or "TIGIT" refers to any native TIGIT polypeptide from any vertebrate origin (including mammals, e.g., primates (e.g., humans and cynomolgus monkeys)), or any fragment thereof, and may optionally contain at most one, at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, at most nine, or at most ten amino acid substitutions, additions, and / or deletions. The term covers full-length, unprocessed TIGIT and any form of TIGIT produced by processing in cells. The term further covers naturally occurring variants of TIGIT, such as splice variants or allelic variants. Non-limiting examples of human TIGIT amino acid sequences targeted by the anti-TIGIT antibodies of the present disclosure are as follows:

[0047] [ka]

[0048] The term "ECD of TIGIT" refers to the extracellular domain of TIGIT. For example, the ECD of the exemplary TIGIT protein set forth in SEQ ID NO: 221 comprises the following amino acid sequence:

[0049] [ka]

[0050] The terms "anti-TIGIT antibody" and "antibody that binds to TIGIT" refer to an antibody that can bind to TIGIT with sufficient affinity so that it can be used as a diagnostic and / or therapeutic agent targeting TIGIT. In one example, the extent of binding of an unrelated, non-TIGIT protein to an anti-TIGIT antibody is less than about 10% of the binding of the antibody to TIGIT, e.g., as measured by BIACORE. (登録商標)In some embodiments, antibodies that bind to TIGIT have a binding affinity of < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -12 M, e.g., 10 -9 M to 10 -10 In some embodiments, the anti-TIGIT antibody binds to a TIGIT epitope that is conserved among TIGITs from different species. In some embodiments, the anti-TIGIT antibody binds to an epitope on TIGIT in the ECD of the protein.

[0051] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In some examples, the chimeric antibodies disclosed herein comprise a camelid heavy chain variable region and a human Fc region.

[0052] As used herein, "CDR" or "complementarity determining region" refers to discontinuous antigen-binding sites within the variable regions of heavy and / or light chains. These specific regions have been described by Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol., 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), in which overlapping or subsets containing amino acid residues are defined when compared with one another. However, it is intended that CDRs referring to antibodies, grafted antibodies, or variants thereof using any one definition be within the scope of the term as defined and used herein. The amino acid residues covering the CDRs defined in each of the above references are listed in Table 1 below for comparison. CDR prediction algorithms and interfaces are known in the art and include, for example, Abhinandan and Martin, Mol. Immunol. [Molecular Immunology], 45:3832-3839 (2008), Ehrenmann F. et al., Nucleic Acids Res. [Nucleic Acids Research], 38:D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res. [Nucleic Acids Research], 43:D432-D438 (2015).The contents of the references referred to in this section are incorporated by reference in their entirety, may be used herein, and may be included in one or more claims herein.

[0053] [Table 1]

[0054] The phrases "variable domain residue numbering, e.g., according to Kabat" or "amino acid position numbering, e.g., according to Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the antibody assembler of Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may have fewer or additional amino acids corresponding to a shortening or insertion of a variable domain FR or CDR. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (based on Kabat residue 52a) and inserted residues after heavy chain FR residue 82 (e.g., based on Kabat residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by comparing the antibody sequence to a "standard" Kabat numbered sequence at the regions of homology.

[0055] In some examples, the amino acid residues covering the CDRs of a single domain antibody (e.g., a single domain anti-TIGIT antibody disclosed herein) are defined according to the IMGT nomenclature of Lefranc et al., supra. In some examples, the amino acid residues covering the CDRs of a full-length antibody are defined according to the Kabat nomenclature of Kabat et al., supra. In some examples, the residue numbering in an immunoglobulin heavy chain, e.g., an Fc region, is that of the EU index as set forth in Kabat et al., supra. The "EU index as set forth in Kabat" is the residue numbering of a human IgG1 EU antibody.

[0056] "Framework" or "FR" refers to those variable domain residues other than the CDR residues as herein defined.

[0057] A "humanized" antibody refers to a chimeric antibody with amino acid residues from non-human CDRs / HVRs or human FRs. In some embodiments, a humanized antibody comprises essentially all of at least one, typically two, variable domains, in which all or essentially all of the HVRs / CDRs correspond to those of a non-human antibody and all or essentially all of the FRs correspond to those of a human antibody. A humanized antibody optionally comprises at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0058] A "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been prepared by any of the techniques disclosed herein for the preparation of human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies may be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals (e.g., immunized xenomouse) that have been modified to produce such antibodies in response to antigen challenge, but in which the endogenous gene locus has been disabled. (商標)(See U.S. Patent Nos. 6,075,181 and 6,150,584 for related technology.) For human antibodies produced by human B cell hybridoma technology, see further, e.g., Li et al., Proc. Natl. Acad. Sci. USA [Publication of the National Academy of Sciences of the United States of America] 103:3557-3562 (2006).

[0059] "Percent (%) amino acid sequence identity" or "homology" between a polypeptide and an antibody sequence identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the polypeptide being compared, after alignment of the sequences (taking into account any conservative substitutions as part of sequence identity). For purposes of determining percent amino acid sequence identity, comparisons can be performed using a variety of methods within the skill of the art, including publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring comparisons, including any algorithm that maximizes comparison over the entire length of the sequences being compared. However, for purposes of this specification, the sequence comparison computer program MUSCLE is used to generate percent amino acid sequence identity values ​​(Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0060] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, when a position in each of two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences, divided by the number of positions compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Comparisons are usually performed when two sequences are aligned to maximize homology.

[0061] The term "constant domain" refers to a portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin, the variable domain, and that contains the antigen-binding site. The constant domain is H 1. C H 2 and C H 3 Domains (C H ) and light chain C L domain.

[0062] The "light chains" of any antibody (e.g., immunoglobulin) of any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), depending on the amino acid sequence of their constant domains.

[0063] The "CH1 domain" (also called "C1" for "H1" domain) is typically from about amino acid 118 to about amino acid 215 (EU numbering system).

[0064] The "hinge region" is usually defined as the region of IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the S—S bonds between the heavy chains in the same positions.

[0065] The "CH2 domain" of the human IgG Fc region (also called the "C2" domain) typically spans from about amino acid 231 to about amino acid 340. The CH2 domain is unique because it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains in intact, native IgG molecules. Speculation suggests that the carbohydrates may provide an alternative for domain-domain pairing and contribute to the stabilization of the CH2 domain. Burton, Molec Immunol., 22:161-206 (1985).

[0066] The "CH3 domain" (also called the "C2" domain) comprises the CH2 domain and residues between the C-terminus of the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence), usually at amino acid residue 446 or 447 of an IgG.

[0067] As used herein, the terms "Fc region" or "fragment crystallizable region" are used to define the C-terminal region of an immunoglobulin heavy chain, and include native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is typically defined as stretching from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus. For example, the C-terminal lysine of the Fc region (based on residue 447 in the EU numbering system) can be removed during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a complete antibody composition may include antibodies with all K447 residues removed, antibodies with K447 residues removed, and mixtures of antibodies with or without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0068] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants or splice forms of these variants. FcγRII receptors include FcγRIIA ("activating receptors") and FcγRIIB ("inhibiting receptors"), which have similar amino acid sequences and are primarily distinguished by their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" herein covers other FcRs and includes FcRs identified in the future.

[0069] As used herein, the term "epitope" refers to the specific atom or amino acid group on an antigen to which an antigen or antigen-binding moiety binds. Two antibodies or antigen-binding moieties can bind to the same epitope in an antigen if they have competitive binding to the antigen.

[0070] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as the binding of a target to an antibody or antibody portion, which determines the presence of the target in the presence of heteromolecules (including biomolecules). For example, an antibody or antibody portion that specifically recognizes a target (which may be an epitope) is an antibody or antibody portion that binds to that target with a longer affinity, avidity, readiness, and / or duration than binding to other targets. In some examples, the extent of binding of an antibody to an unrelated target is about 10% less than the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some examples, the dissociation constant (K) of an antibody that specifically binds to a target is about 10% less than the extent of binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). D )≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, this may include, but is not limited to, exclusive binding. The binding specificity of an antibody or antigen-binding domain may be determined experimentally by methods known in the art. Such methods include Western blot, ELISA, RIA, ECL, IRMA, EIA, BIACORE. TM -Including but not limited to testing and peptide scanning.

[0071] An "isolated" antibody (or construct) is an antibody that has been identified, isolated, and / or recovered from a component (e.g., natural or recombinant) of its production environment. In some embodiments, an isolated polypeptide is free or essentially free from association with all other components in its production environment.

[0072] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminant nucleic acid molecule normally associated with it in its production environment. In some embodiments, an isolated nucleic acid is free from, or substantially free from, all components associated with the production environment. The form of the isolated nucleic acid molecule encoding the polypeptides and antibodies described herein differs from its naturally occurring form or background. Thus, an isolated nucleic acid molecule differs from a nucleic acid encoding the polypeptides and antibodies described herein that is naturally present in a cell. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0073] The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences applicable to prokaryotes include promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0074] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to DNA for a coding sequence if it affects the transcription of the sequence; or a ribosomal binding site is operably linked to a coding sequence if it is positioned favorably for translation. Typically, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0075] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which they are introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0076] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid, including the primary target cell and its progeny.

[0077] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of transfers. The nucleic acid content of the progeny may differ from that of the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0078] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the subject is a human.

[0079] An "effective amount" of a drug refers to an amount that effectively achieves a desired therapeutic or prophylactic effect at a required dosage and for a required period of time. The specific dosage may vary depending on one or more of the particular drug selected, the subsequent administration regimen (whether or not combined with other compounds), the time of administration, the tissue imaged, and the physical delivery system associated therewith.

[0080] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist of the present application may vary depending on factors such as, for example, the disease state, age, sex, and weight of the individual and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the substance / molecule, agonist, or antagonist are counteracted by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations.

[0081] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, such a prophylactically effective amount will be less than the therapeutically effective amount.

[0082] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results (including clinical results). For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the worsening of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or slowing the rate of disease progression, ameliorating the disease state, providing partial or complete relief from the disease, reducing the dosage of one or more other drugs required to treat the disease, slowing the progression of the disease, increasing or improving quality of life, increasing weight gain, and / or prolonging survival. "Treatment" further covers reducing the pathological consequences of cancer (e.g., tumor volume). The methods of the present application contemplate any one or more of these aspects of treatment. "Treatment" does not necessarily mean that the disease being treated is cured.

[0083] It is to be understood that the examples of the application described herein include "consisting of" and "consisting essentially of."

[0084] As used herein, "about" or "approximately" means that a particular value, as determined by one of ordinary skill in the art, is within an acceptable error range, which depends in part on how the value is measured or determined, i.e., is limited by the measurement system. In some embodiments, "about" may mean within three or more standard differences, in accordance with practice in the art. In some embodiments, "about" can refer to a range of at most 20% (e.g., at most 10%, at most 5%, or at most 1%) of a given value. In some embodiments, particularly with respect to biological systems and methods, the term may mean within an order of magnitude, e.g., within 5-fold or 2-fold, of a value.

[0085] As used herein, the term "modulation" refers to a change in a positive or negative direction. Exemplary modulations include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0086] As used herein, the term "increase" refers to a change in a positive direction of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0087] As used herein, the term "decrease" refers to a change in the negative direction of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0088] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0089] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0090] "Effector functions" refer to those biological activities of the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0091] An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules (including, but not limited to, cytotoxic agents).

[0092] The term "drug formulation" is a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain other ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0093] As used herein, a "pharmaceutically acceptable carrier agent" refers to an ingredient in a drug formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carrier agents include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0094] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is responsible for binding the antibody to an antigen. In some embodiments, the heavy and light chain variable domains (VH and VL, respectively) of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FR) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 61st ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, VH or VL domains can be used to isolate antibodies that bind to a specific antigen from those that do, and to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0095] 2. Antibodies In some embodiments, the present invention is based in part on the discovery of single domain antibodies that bind to TIGIT, which can be used in anti-tumor therapy, where the antibodies selectively inhibit the TIGIT receptor and induce beneficial immune responses in immune cells (e.g., T cells). Accordingly, the present disclosure provides anti-TIGIT antibodies. In some embodiments, the anti-TIGIT antibodies disclosed herein are antagonistic antibodies that inhibit TIGIT receptor function. In some embodiments, the anti-TIGIT antibodies block the interaction between the TIGIT receptor and its ligand. In some embodiments, the anti-TIGIT antibodies block immune inhibitory signals from the TIGIT receptor. In some embodiments, the anti-TIGIT antibodies include single domain antibodies, such as camelid antibodies or VHH antibodies. In some embodiments, the anti-TIGIT antibodies have improved tissue penetration capabilities due to their smaller size compared to conventional antibodies in IgG, Fab, and / or scFv formats.

[0096] In some embodiments, the antibodies of the present disclosure may be or include monoclonal antibodies (including chimeric, humanized, or human antibodies). In some embodiments, the antibodies disclosed herein include humanized antibodies. In some embodiments, the antibodies include a covalent human framework, e.g., a human immunoglobulin framework or a human covalent framework.

[0097] In some embodiments, an antibody of the present disclosure may be an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In some embodiments, the antibody is a full-length antibody, e.g., a complete IgG1 antibody, or other antibody type or isotype as defined herein. In some embodiments, an antibody of the present disclosure may incorporate any of the features described in this application (e.g., Sections 2.1-2.11, detailed herein), either singly or in combination.

[0098] The antibodies of the present disclosure can be used, for example, to diagnose or treat neoplasia or cancer. In some embodiments, tumorigenesis and cancers whose growth can be inhibited using the antibodies of the present disclosure include tumorigenesis and cancers that typically respond to immunotherapy. In some embodiments, tumorigenesis and cancers include breast cancer (e.g., mammary gland cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), and renal cell carcinoma (RCC). Other examples of cancers that can be treated with the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma, lymphocytic lymphoma, primary central nervous system (CNS) lymphoma, T-cell lymphoma), nasopharyngeal carcinoma, head or neck cancer, skin cancer or intraocular malignant melanoma, uterine cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, external vagina, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, and paramammary gland cancer. gland), soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, breast cancer, pelvic cancer, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including cancers induced by asbestos (e.g., mesothelioma), and combinations of the above cancers.

[0099] 2.1 Exemplary Anti-TIGIT Antibodies The present disclosure provides isolated antibodies that bind to TIGIT protein. In some embodiments, the anti-TIGIT antibodies of the present disclosure bind to the ECD of TIGIT. In some embodiments, the anti-TIGIT antibodies bind to the ECD of TIGIT comprising the amino acid sequence set forth in SEQ ID NO: 222. In some embodiments, the anti-TIGIT antibodies bind to the same epitope as the anti-TIGIT antibodies described herein (e.g., 2A3).

[0100] In some embodiments, the anti-TIGIT antibodies disclosed herein may be used as antagonists of the TIGIT receptor. In some embodiments, the anti-TIGIT antibodies can reduce TIGIT receptor activity by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9%. In some embodiments, the anti-TIGIT antibodies can block immune inhibitory signaling downstream of the TIGIT receptor. In some embodiments, the anti-TIGIT antibodies increase the immune response and / or anti-tumor activity of immune cells (e.g., T cells and / or NK cells). In some embodiments, treatment with the anti-TIGIT antibodies shows anti-tumor effects in test subjects, thereby reducing tumor growth and / or prolonging the subject's survival. In some embodiments, anti-TIGIT antibodies, including single-domain antibodies (e.g., VHHs), have a smaller molecular size than full-length antibodies because the size of single-domain antibodies is smaller than the Fab domain of full-length antibodies, and thus can induce superior tissue penetration (e.g., at tumor sites) compared to full-length antibodies. In some embodiments, treatment with the anti-TIGIT antibodies exhibits a superior anti-tumor effect compared to treatment with full-length anti-TIGIT antibodies (e.g., reference Ab1 and reference Ab2). Reference Ab1 has the same amino acid sequence as BMS 22G2, which is disclosed in US2016 / 0176963 A1, and reference Ab2 has the same amino acid sequence as tiragolumab, which is disclosed in US2017 / 0088613 A1.

[0101] In some embodiments, the anti-TIGIT antibody comprises a single domain antibody that binds to TIGIT. In some embodiments, the single domain antibody comprises a VHH. In some embodiments, the single domain antibody comprises a heavy chain variable region (VH). In some embodiments, the single domain antibody is linked to an Fc region. In some embodiments, the single domain antibody is not linked to an Fc region.

[0102] In some embodiments, the single domain antibody is about 1 x 10 -7 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 1×10 -8 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 5×10 -9 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 1×10 -9 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 1×10 -9 M to approximately 1 x 10 -7 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 1×10 -9 M to approximately 1 x 10 -8 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 2×10 -9 M to approximately 1 x 10 -8 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 2×10 -9 M to about 5 x 10 -8 In some embodiments, the single domain antibody binds to TIGIT with a KD of about 1×10 -9 M to about 5 x 10 -9 Binds to TIGIT via the KD of M.

[0103] In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 96. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 98, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 99, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 100. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 102, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 103, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 104. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 106, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 107, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 108. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 110, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 111, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 112.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 114, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 115, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 116. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 118, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 119, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 120. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 122, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 123, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 124. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 126, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 127, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 128. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 130, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 136. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 138, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 139, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 140. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 142, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 143, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 146, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 147, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 148. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 150, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 151, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 152.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 154, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 156. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 158, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 159, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 160. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 162, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 163, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 164. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 166, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 168. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 170, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 171, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 172.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 174, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 175, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 176. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 178, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 182, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 183, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 184. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 186, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 187, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 188. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 190, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192.

[0104] In some embodiments, the single domain antibody comprises a heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, and 190, or a variant thereof having up to about three amino acid substitutions; and b) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, and 190, or a variant thereof having up to about three amino acid substitutions. and c) a heavy chain variable region CDR2 comprising any one of the amino acid sequences of SEQ ID NOs: 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, and 192, or a variant thereof having up to about three amino acid substitutions.

[0105] In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain, and the CDR3 domain respectively comprise the CDR1 domain, the CDR2 domain, and the CDR3 domain comprise the CDR1 domain, the CDR2 domain, and the CDR3 domain comprise the CDR3 domain contained in a reference heavy chain variable region, and the reference heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, and 193.

[0106] In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 96. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 98, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 99, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 100. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 102, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 103, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 104. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 106, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 107, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 108. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 110, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 111, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 112. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 114, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 115, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 116. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 118, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 119, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 120.In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 122, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 123, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 124. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 126, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 127, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 128. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 130, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 131, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 132. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 136. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 138, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 139, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 140. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 142, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 143, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 144. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 146, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 147, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 148.In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 150, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 151, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 152. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 154, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 155, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 156. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 158, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 159, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 160. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 162, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 163, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 164. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 166, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 167, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 168. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 170, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 171, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 172. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 174, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 175, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 176.In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 178, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 182, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 183, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 184. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 186, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 187, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 188. In some embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 190, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 192.

[0107] In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, and 193. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, and 193.

[0108] In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 97. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 101. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 105. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 117. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 121. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 125. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 133. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 137. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 141. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 145. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 149. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 153. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 157.In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 161. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 165. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 169. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 177. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 181. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 185. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 189. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 193.

[0109] In some embodiments, any amino acid sequence contained in the heavy chain variable region may contain at most about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the amino acid substitutions are conservative substitutions.

[0110] In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 5, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 6, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 7. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 11. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 13, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 14, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 17, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 18, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 19.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 23. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 25, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 26, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 27. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 29, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 30, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 31. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 33, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 34, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 35. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 37, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 38, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 39.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 45, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 46, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 47. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 49, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 50, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 51. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 53, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 54, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 55. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 57, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 58, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 59.In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 63. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 65, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 66, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 67. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 69, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 70, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 71. In some examples, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 74, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 75. In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 77, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 78, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 79.In some embodiments, the single domain antibody cross-competes with a reference anti-TIGIT single domain antibody for binding to TIGIT, the reference anti-TIGIT single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 81, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 82, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 83.

[0111] In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 5, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 6, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 7. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 9, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 10, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 11. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 13, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 14, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 15. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 17, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 18, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 19. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 23.In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 25, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 26, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 27. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 29, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 30, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 31. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 33, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 34, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 35. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 37, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 38, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 39. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 45, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 46, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 47.In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 49, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 50, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 51. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 53, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 54, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 55. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 57, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 58, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 59. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 63. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 65, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 66, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 67. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 69, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 70, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 71.In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 74, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 75. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 77, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 78, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 79. In some embodiments, the single domain antibody comprises a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 81, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 82, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 83.

[0112] In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, and 84. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, and 84.

[0113] In some embodiments, any amino acid sequence contained in the heavy chain variable region may contain at most about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 amino acid substitutions, deletions, and / or additions. In some embodiments, the amino acid substitutions are conservative substitutions.

[0114] In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 64.In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 72. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 76. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the single domain antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 84.

[0115] In some embodiments, the single domain antibody comprises a humanized framework. In some embodiments, the humanized framework comprises a framework sequence of a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 85-93. In some embodiments, the humanized framework comprises a FR2 sequence of a heavy chain variable region sequence selected from the group consisting of SEQ ID NOs: 85-93.

[0116] In some embodiments, the anti-TIGIT antibody does not comprise an Fc region. In some embodiments, the anti-TIGIT antibody comprises an Fc region. In some embodiments, the Fc region comprises a human Fc region. In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In some embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In some embodiments, the Fc region comprises an IgG1 Fc region. In some embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, Y300L, and P396L. In some embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E. In some embodiments, the anti-TIGIT antibody comprises the amino acid sequence set forth in SEQ ID NO:194.

[0117] In some embodiments, the heavy chain variable region is linked to the Fc region via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker comprises about 4 to about 30 amino acids. In some embodiments, the peptide linker comprises about 4 to about 15 amino acids. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 195-220.

[0118] In some embodiments, the anti-TIGIT antibody comprises a multispecific antibody, e.g., a bispecific antibody, a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof. In some embodiments, the antibody comprises a multispecific antibody (e.g., a bispecific antibody), which comprises a second antibody portion that specifically binds to a second antigen.

[0119] In some embodiments, the second antigen is a tumor-associated antigen. In some embodiments, the tumor-associated antigen is selected from the group consisting of Her-2, EGFR, PD-L1, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein ( EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insert domain receptor (KDR), Lewis A (CA1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, carcinoembryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), inclusion protein 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), tyrosine kinase transmembrane receptor 1 (ROR1), and any combination thereof.

[0120] In some embodiments, the second antigen is an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator is selected from the group consisting of PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof.

[0121] In some embodiments, the anti-TIGIT antibody is conjugated to a therapeutic agent or marker. In some embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0122] 2.2 Antibody affinity In some embodiments, the antibodies or antigen-binding portions of the multispecific antibodies disclosed herein have high binding affinity for their target antigens. In some embodiments, the antibodies or antigen-binding portions have a binding affinity of about 1×10 -7 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 1×10 -8 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 5×10 -9 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 1×10 -9 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 1×10 -9 M to approximately 1 x 10 -7 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 1×10 -9 M to approximately 1 x 10 -8 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 2×10 -9 M to approximately 1 x 10 -8 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 2×10 -9 M to about 5 x 10 -8 In some embodiments, the antibody or antigen-binding portion binds to the target with a KD of about 1×10 -9 M to about 5 x 10 -9 It binds to the target with a KD of M.

[0123] The KD of an antibody or antigen-binding portion may be determined by methods known in the art, such as Western blot, ELISA, RIA, ECL, IRMA, EIA, Octet-BIACORE (登録商標) -Including but not limited to testing and peptide scanning.

[0124] In some embodiments, BIACORE (登録商標)Surface plasmon resonance assays may be used to measure KD, for example, on immobilized antigen CMS chips at approximately 10 response units (RU) at 25°C using a BIACORE™. (登録商標) -2000 or BIACORE (登録商標) Measurements are performed using, but not limited to, a Biacore 3000 (Biacore, Piscataway, NJ). In some examples, a carboxymethylated dextran synthesizer sensor chip (CMS, Biacore) is activated using N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) using 10 mM sodium acetate, pH 4.8, and injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of the conjugated protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) are injected at a flow rate of approximately 25 μl / min at 25° C. The association rate (k on ) and dissociation rate (k off ) is based on a simple one-to-one Langmuir binding model (BIACORE (登録商標) The equilibrium dissociation constant (KD) may be calculated as the ratio k0fffkon using the evaluation software version 3.2. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate measured by surface plasmon resonance assay is 10 6 M -l s -1 Above this, the association rate may be determined by a fluorescence quenching technique, which is performed with increasing antigen concentration (e.g., a spectrophotometer, e.g., a spectrophotometer in a pass-cut configuration (Afaff Instruments) or an 8000 series SLM-AMINCO with a stirred absorbent pool). (商標)The increase or decrease in fluorescence emission intensity (excitation = 295 nM, emission = 340 nM, 16 nM bandpass) of 20 nM anti-antigen antibody (Fab format) in PBS (pH 7.2) at 25°C is measured in the presence of a spectrophotometer (measured by ThermoSpectronic).

[0125] 2.3 Antibody fragments In some embodiments, antibodies of the present disclosure include antigen-binding fragments or antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab'), Fv, and scFv fragments, as well as other fragments described below. For a review of several antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthin, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab)2 fragments that contain rescued receptor binding epitope residues and have increased in vivo half-lives.

[0126] In some embodiments, the antibodies of the present disclosure may be diabodies. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are further described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0127] In some embodiments, antibodies of the present disclosure may include single domain antibodies. Single domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, the single domain antibody is a human single domain antibody (Domantis, Waltham, Massachusetts (MA); see, e.g., U.S. Pat. No. 6,248,516 B1). In some embodiments, the single domain antibody is a camelid single domain antibody. In some embodiments, the single domain antibody is a VHH. In some embodiments, the single domain antibody is humanized.

[0128] Antibody fragments may be prepared by several techniques, including, but not limited to, proteolytic digestion of whole antibodies and recombinant host cell (e.g., E. coli or phage) production, as described herein.

[0129] 2.4 Chimeric and humanized antibodies In some embodiments, an antibody comprising an antigen-binding portion of a multispecific antibody of the present disclosure is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In some embodiments, a chimeric antibody is a "class-switched" antibody, in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0130] In some examples, an antibody comprising an antigen-binding portion of a multispecific antibody of the present disclosure may be a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains, in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody, and the FRs (or any portions thereof) are derived from human antibody sequences. Optionally, the humanized antibody may further comprise at least a portion of a human constant region. In some examples, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody derived from the HVR residues), e.g., to restore or improve antibody specificity or affinity.

[0131] Humanized antibodies and methods for their preparation are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988), and in Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (SDR (a-CDR) grafting described); Padlan, Mol. Immunol. 28:489-498 (1991) (where "surface remodification" is described), Dall'Acqua et al., Methods 36:43-60 (2005) (where "FR shuffling" is described), Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (where a "guided selection" method for FR shuffling is described).

[0132] Human framework regions that can be used for humanization include framework regions selected by the "best-fit" method (e.g., Sims et al., J. Immunol. 1512296 (1993)), framework regions derived from shared sequences of human antibodies of a particular subclass of light or heavy chain variable region (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and Presta et al., J. Immunol. 151:2623 (1993)), framework regions derived from human mature (somatically transformed) antibodies, and framework regions derived from human antibodies of a particular subclass of light or heavy chain variable region (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and Presta et al., J. Immunol. 151:2623 (1993)). These framework regions include, but are not limited to, human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0133] 2.5 Human antibodies In some embodiments, the antibodies of the present disclosure may be human antibodies (e.g., human domain antibodies or human DAbs). Human antibodies may be produced using different techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. [Current Trends in Pharmacology] 5:368-74 (2001); Lonberg, Curr. Opin. Immunol. [Current Trends in Immunology] 20:450-459 (2008); and Chen, Mol. Immunol. [Molecular Immunology] 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single domain antibodies (or DAbs) are known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0134] Human antibodies (e.g., human DAbs) can be prepared by administering an immunogen to transgenic animals that have been modified to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Further, see, e.g., XENOMOUSE. TM US Patent Nos. 6,075,181 and 6,150,584, describing the technology, HuMab (登録商標) U.S. Patent No. 5,770,429, KM MOUSE, describes the technology (登録商標) US Patent No. 7,041,870 describing the technology, and VelociMouse (登録商標) See US Patent No. US2007 / 0061900, which describes the technology. The human variable regions from whole antibodies produced by such animals may be further modified (e.g., by attaching to different human constant regions).

[0135] Human antibodies (e.g., human DAbs) may be prepared using hybridoma-based methods. Human myeloma and murine human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, e.g., Kozbor J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987; and Boerner et al., J. Immunol. 147:86 (1991)). Li et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006) further describe human antibodies produced by human B-cell hybridoma technology. Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue [Modern Immunology], 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0136] Human antibodies (e.g., human DAbs) may be generated by isolating Fv clone variable domain sequences selected from a human phage display library. These variable domain sequences may then be combined with the necessary human constant domains. A description of techniques for selecting human antibodies from antibody libraries follows.

[0137] 2.6 Library-derived antibodies Antibody portions can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, several methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, eds., Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003; Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004). Methods for constructing single domain antibody libraries have been described; see, e.g., U.S. Pat. No. 7,371,849.

[0138] In some phage display methods, V H and V LGene libraries can be cloned individually, randomly recombined into phage libraries, and screened for antigen-binding phages as described in Winter et al., Ann. Rev. Immunol. [Annual Comments on Immunology], 12:433-455 (1994). Phages typically display antibody fragments as scFv or Fab fragments. Libraries from immune sources can provide high-affinity antibodies to immunogens without constructing hybridomas. Alternatively, natural libraries (e.g., obtained from humans) can be cloned without the need for any immunization, providing a single source of antibodies against a wide range of non-self and self antigens, as described in Griffiths et al., EMBO J. [European Journal of Molecular Biology], 12:725-734 (1993). Finally, natural libraries can be synthesized by cloning unrearranged V gene fragments from cells, using PCR primers containing random sequences to encode highly variable CDR3 regions, and completing the reassortment in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include U.S. Patent No. 5,750,373 and U.S. Patent Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0139] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0140] 2.7 Antibody variants The presently disclosed subject matter further provides amino acid sequence variants of the disclosed antibodies. For example, improvements in the binding affinity and / or other biological properties of the antibody may be required. Amino acid sequence variants of the antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, but are not limited to, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final (i.e., modified) antibody possesses the required properties (e.g., antigen binding).

[0141] 2.7.1 Substitution, Insertion, and Deletion Mutants In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 2 and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for the desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0142] [Table 2]

[0143] Amino acids may be grouped according to common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln, (3) acidic: Asp, Glu, (4) alkaline: His, Lys, Arg, (5) residues that influence chain orientation: Gly, Pro, and (6) aromatic: Trp, Tyr, Phe. In some embodiments, non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0144] In some embodiments, one type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). The resulting variants are typically selected for further study to modify (e.g., improve) some biological property (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and / or essentially retain some biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which is easy to generate, e.g., using phage-display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR (or CDR) residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0145] Modifications (e.g., substitutions) can be made in HVRs (or CDRs) to, for example, improve antibody affinity. Such modifications can be made in HVR (or CDR) "hot spots" (i.e., residues encoded by codons frequently mutated during the somatic maturation process) (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or in SDRs (a-CDRs), and the resulting variant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any one of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis), generating a secondary library. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves HVR (or CDR)-directed methods, in which a few HVR (or CDR) residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify HVR (or CDR) residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are routinely targeted.

[0146] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs (or CDRs), so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions according to the present specification) may be made in an HVR (or CDR) that do not substantially reduce binding affinity. Such modifications may be in HVR (or CDR) "hot spots" or outside of a CDR. In some embodiments of the variant VHH sequences provided above, each HVR (or CDR) is unaltered or contains one, two, or more than three amino acid substitutions.

[0147] As described in Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying antibody residues or regions amenable to targeted mutagenesis is called "alanine scanning mutagenesis." In such methods, a target residue or group of residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) is identified, and substitution with neutral or negatively charged amino acids (e.g., alanine or polyalanine) is performed to determine whether the antibody-antigen interaction is affected. Further substitutions at the amino acid position can be introduced to demonstrate functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as substitution candidates. Mutants can be screened to determine whether they contain the desired attributes.

[0148] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0149] 2.7.2 Glycosylation variants In some embodiments, the antibody portion is modified to increase or decrease the degree of glycosylation of the construct. Addition or deletion of glycosylation sites to an antibody can be readily accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0150] When the antibody moiety includes an Fc region (e.g., scFv-Fc), the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched, high-contact angle oligosaccharides, which are typically N-linked to the Fc region C. H The oligosaccharide is linked to Asn297 of the .2 domain. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, and fucose attached to the GlcNAc in the "stem" of the high-contact-angle oligosaccharide. In some embodiments, modifications can be made to the oligosaccharide in the antibody moiety to generate antibody variants with improved properties.

[0151] In some embodiments, the antibody portion comprises a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the sum of all glycostructures (e.g., complex, heterozygous, and high-mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue at about position 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence variation in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function (see, e.g., US Patent Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.)). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J. Mol. Biol. [Journal of Molecular Biology] 336:1239-1249 (2004), Yamane-Ohnuki et al. Biotech. Bioeng. [Biotechnology and Bioengineering] 87:614 (2004).An example of a cell line capable of producing defucosylated antibodies is the protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. [Biochemistry and Biophysics Publications] 249:533-545 (1986); U.S. Patent No. US 2003 / 0157108). A1, Presta, L, and WO2004 / 056312A1, Adams et al.), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. [Biotechnology and Bioengineering] 87:614 (2004), Kanda, Y. et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering], 94(4):680-688 (2006) and WO2003 / 085107).

[0152] In some embodiments, the antibody portion has a bisected oligosaccharide, for example, where a high-contact angle oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Further provided are antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0153] 2.7.3 Fc Region Variants In some examples, Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region of an antibody portion (e.g., scFv-Fc). The Fc region variants may comprise a single human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region), which sequence comprises amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0154] In some embodiments, the Fc fragment possesses some (but not all) effector functions, making it a desirable candidate for applications in which in vivo half-life of the antibody portion is important, but some effector functions (e.g., complement and ADCC) are unnecessary or deleterious. Reduced / depleted CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody does not bind FcγR (and thus may lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. [Annual Comments on Immunology] 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA, 82:1499-1502 (1985), 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med., 166:1351-1361 (1987)). Alternatively, non-radioactive assays (e.g., ACTI for flow cytometry) can be used. (商標) Non-radioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, CA, and CytoTox 96) (登録商標)Non-radioactive cytotoxicity assays (see Promega, Madison, Wisconsin) may also be used. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule may be assessed in vivo, for example, in an animal model, as described, for example, in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0155] Antibodies with reduced effector function (U.S. Pat. No. 6,737,056) include antibodies with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329. Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0156] Described herein are several antibody variants with improved or decreased binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In some embodiments, the Fc fragment is an IgG1 Fc fragment. In some embodiments, the IgG1 Fc fragment comprises an L234A mutation and / or an L235A mutation. In some embodiments, the Fc fragment is an IgG2 or IgG4 Fc fragment. In some embodiments, the Fc fragment is an IgG4 Fc fragment comprising an S228P, F234A, and / or L235A mutation.

[0157] In some embodiments, the antibody portion comprises an Fc region with one or more amino acid substitutions, wherein these substitutions (e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) within the Fc region) improve ADCC.

[0158] In some embodiments, modifications occur in the Fc region that result in modified (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 164:4178-4184 (2000).

[0159] In some embodiments, the antibody portion (e.g., scFv-Fc) variant comprises a variant Fc region, which comprises one or more amino acid substitutions that alter half-life and / or binding to the neonatal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), e.g., as described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more amino acid substitutions, where these substitutions alter binding of the Fc region to FcRn. Such Fc variants include those variants with substitutions at one or more Fc region residues (eg, substitution of Fc region residue 434) (US Pat. No. 7,371,826).

[0160] See also Duncan and Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants.

[0161] 2.7.4 Cysteine ​​Engineered Antibody Variants In some examples, this may involve the generation of cysteine-engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In some examples, the substituted residues are located at accessible sites of the antibody. By replacing these residues with cysteine ​​residues, reactive thiol groups are located at accessible sites of the antibody, which may be used to couple the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In some examples, any one or more residues of A118 (EU numbering) of the heavy chain and S400 (EU numbering) of the heavy chain Fc region may be replaced with cysteine ​​residues. Cysteine-engineered antibody moieties may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0162] 2.8 Antibody derivatives In some embodiments, the antibody moieties described herein may be further modified to include other non-proteinaceous moieties known in the art and readily available. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) with dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in formulations due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one type of polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used for a determined diagnostic condition, etc.

[0163] In some embodiments, the antibody portion may be further modified to include one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biologically active" or "having biological activity" refers to a biological activity exhibited in vivo to perform a specific function. For example, it may mean binding to a specific biological molecule (e.g., protein, DNA, etc.) and promoting or inhibiting the activity of such a biological molecule. In some embodiments, biologically active proteins or fragments thereof include proteins or polypeptides administered to a patient as active drug substances, proteins and polypeptides for purposes of prevention or treatment and diagnosis of disease or conditions (e.g., enzymes used in diagnostic tests or in vitro assays), and proteins and polypeptides administered to a patient to prevent disease (e.g., vaccines).

[0164] 2.9 Antibody Production Methods Any available or known technique in the art can be used to produce the antibodies disclosed herein. For example, but not limited to, the antibodies can be produced using the recombinant methods and compositions described in U.S. Patent No. 4,816,567. Detailed steps for producing the antibodies are described in detail in the examples below.

[0165] The presently disclosed subject matter further provides isolated nucleic acids encoding the antibodies disclosed herein. For example, the isolated nucleic acids can encode an amino acid sequence comprising the VL and / or the VH of the antibody, e.g., the light chain and / or the heavy chain of the antibody.

[0166] In some embodiments, the nucleic acid may be present in one or more vectors (e.g., expression vectors). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which can have another DNA segment ligated into a circular double-stranded DNA loop therein. Another type of vector is a viral vector, in which another DNA segment can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and thereby replicate along with the host genome. Additionally, some vectors, such as expression vectors, can direct the expression of genes to which they are operably linked. Generally, expression vectors used in recombinant DNA techniques are always in the form of plasmids (vectors). However, the disclosed subject matter is intended to include other expression vectors that serve equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0167] Different portions of the antibodies disclosed herein can be constructed in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Foot and Mouth Disease Virus IRES, Picornales IRES, Poliovirus IRES, and Encephalomyocarditis Virus IRES), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Retroviral vectors in combination with appropriate packaging threads are also suitable, in which the capsid protein is capable of infecting human cells. Cell lines producing various amphipathic viruses are known and include, but are not limited to, PA12 (Miller et al. (1985) Mol. Cell. Biol. 5:431-437), PA317 (Miller et al. (1986) Mol. Cell. Biol. 6:2895-2902), and CRIP (Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Nonamphipathic particles are also suitable, for example, using VSVG, RD114, or GALV envelopes and any other pseudotyped particles known in the art.

[0168] In some embodiments, nucleic acids encoding an antibody of the present disclosure and / or one or more vectors comprising the nucleic acids can be introduced into a host cell. In some embodiments, nucleic acids can be introduced into cells by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or phage vector comprising the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In some embodiments, the host cell can include, for example, a host cell transformed with a vector comprising a nucleic acid encoding a single domain antibody and / or an amino acid sequence comprising a VH of the single domain antibody. In some embodiments, the host cell can include, for example, a host cell transformed with (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antibody and an amino acid sequence comprising a VH of the antibody, or (2) a first vector comprising a nucleic acid encoding the amino acid sequence of the VL of the antibody and a second vector comprising a nucleic acid encoding the amino acid sequence of the VH of the antibody. In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, YO, NSO, Sp20 cell).

[0169] In some embodiments, methods for preparing the antibodies disclosed herein may include culturing host cells into which nucleic acid encoding the antibody has been introduced under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells and / or host cell culture medium. In some embodiments, the antibody is recovered from the host cells by chromatographic techniques.

[0170] To recombinantly produce the antibodies of the present disclosure, nucleic acids encoding the antibodies described above can be isolated, inserted into one or more vectors, and further cloned and / or expressed in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains). Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic and eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when fucosylation and Fc effector functions are not required. See, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 for information on expressing antibody fragments and polypeptides in bacteria. (See further the expression of antibody fragments in E. coli as described in Charlton, Methods in Molecular Biology, Vol. 248 (ed. by BKC Lo, Human Press, Totowa, NJ, 2003), pp. 245-254.) Following expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0171] In addition to prokaryotes, eukaryotic microbes (e.g., filamentous fungi or yeast) are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gemgross, Nat. Biotech. [Natural Biotechnol.] 22:1409-1414 (2004) and Li et al., Nat. Biotech. [Natural Biotechnol.] 24:210-215 (2006). Suitable host cells for expressing glycosylated antibodies may be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrates include plants and insect cells. Many baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. In some embodiments, plant cell cultures may be used as host cells. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (PLANTIBODIES FOR PRODUCING ANTIBODIES IN TRANSGENIC PLANTS). (商標) See, for example, the technique described herein.

[0172] In some embodiments, vertebrate cells may be used as host cells, for example, but not limited to, mammalian cell lines adapted for suspension growth may be useful. Non-limiting examples of useful mammalian host cell lines include monkey kidney CV1 transformed with SY40 (COS-7), human embryonic kidney (293 or 293 cells, e.g., those described in Graham et al., J Gen Viral. 36:59 (1977)), baby hamster kidney (BHK) cells, mouse Sertoli cells (TM4 cells, e.g., those described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney (CV1), African midge kidney (VERO-76) cells, human cervical carcinoma (HELA) cells, canine kidney (MDCK) cells, buffalo rat hepatocytes (BRL 3A), human lung (W138) cells, human hepatocytes (Hep 02), mouse mammary tumor (MMT 060562), TRI cells, e.g., those described in Mather et al., Annals

[0004] Examples of useful mammalian host cell lines include those described in NY Acad. Sci. 383:44-68 (1982), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:42 I6 (1980)) and myeloma cell lines (e.g., YO, NSO, and Sp2 / 0). For a review of several mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Human Press, Totowa, NJ), pp. 255-268 (2003).

[0173] In some embodiments, techniques for preparing bispecific and / or multispecific antibodies include, but are not limited to, recombinantly discovering two immunoglobulin heavy chain and light chain pairs with the same specificity, where one or both heavy or light chains are fused to an antigen-binding moiety (e.g., a single domain antibody, e.g., VHH) with a different specificity, resulting in recombinant co-expression of two immunoglobulin heavy chain and light chain pairs with different specificities (see, e.g., Milstein and Cuello, Nature 305:537 (1983)); PCT Patent Application No. WO 93 / 08829; and Traunecker et al., EMBO J 10:3655 (1991)), and "punch and die" engineering (see, e.g., U.S. Pat. No. 5,731,168). Bispecific antibodies can also be prepared by engineering electrostatic deflection effects to form antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004). A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)). Trispecific antibodies are prepared as described in Immunol. 147:60 (1991).

[0174] Bispecific and multispecific molecules of the present disclosure may be prepared by chemical techniques (see, e.g., Kranz (1981) Proc. Natl. Acad. Sci. USA [Publication of the National Academy of Sciences of the United States of America] 78:5807), "polyoma" techniques (e.g., U.S. Pat. No. 4,474,893), or recombinant DNA techniques. Bispecific and multispecific molecules of the presently disclosed subject matter may be prepared by conjugating constituent binding specificities, e.g., a first epitope and a second epitope binding specificity, using methods known in the art and described herein. For example, but not limited to, each binding specificity of the bispecific and multispecific molecule may be prepared together or separately and then conjugated to each other using recombinant fusion protein technology. When the binding specificities are proteins or peptides, covalent conjugation can be achieved using a variety of coupling or cross-linking agents. Non-limiting examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) 78th ed., 118-132; Brennan (1985) Science 229:81-83); Glennie (1987) J. Immunol. 139:2367-2375). When the binding specificities are antibodies (e.g., two humanized antibodies), they may be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In some embodiments, prior to conjugation, the hinge region may be modified to contain an odd number of sulfhydryl residues (e.g., one).

[0175] In some embodiments, the two binding specificities of a bispecific antibody can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific and multispecific molecule is a MAb x MAb, MAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. In some embodiments, the bispecific antibody of the present disclosure can be a single-chain molecule, e.g., a single-chain bispecific antibody, a single-chain bispecific molecule comprising one single-chain antibody and a binding determinant cluster, or a single-chain bispecific molecule comprising two binding determinant clusters. Bispecific and multispecific molecules can be single-chain molecules or can comprise at least two single-chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653, U.S. Patent No. 5,258,498, and U.S. Patent No. 5,482,858. The present specification further includes engineered antibodies with three or more functional antigen binding sites (e.g., epitope binding sites), including "octopus antibodies" (see, e.g., US 2006 / 0025576 A1).

[0176] In some embodiments, an animal system can be used to generate the antibodies of the present disclosure. The animal system for preparing hybridomas is the murine system.

[0177] Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0178] 2.10 Assay The antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by a number of assays known in the art and as described herein.

[0179] In some embodiments, the antigen-binding activity of an antibody of the present disclosure can be tested by known methods (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay). Each of these assays typically detects the presence of a protein-antibody complex of particular interest by a labeled reagent (e.g., an antibody) with specificity for the complex of interest. For example, antibodies can be detected by, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody. Alternatively, the antibody can be detected by any one of several other immunoassays. For example, the antibody can be radiolabeled and used in a radioimmunoassay (RIA). (See, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference.) The radioactive isotope can be detected by such means as the use of, for example, a Geiger counter or a scintillation counter or by autoradiography.

[0180] In some embodiments, competition assays may be used to identify antibodies that compete with an antibody of the present disclosure (e.g., 1C12, 2A3, or 1G1) for binding to TIGIT. In some embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) bound by 1C12, 2A3, or 1G1. Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66 (Human Press, Totowa, NJ) provides detailed exemplary methods for mapping epitopes bound by antibodies.

[0181] In a non-limiting example of a competitive assay, immobilized TIGIT is incubated in a solution containing a first labeled antibody (e.g., 1C12, 2A3, or 1G1) that binds to TIGIT and a second unlabeled antibody, and the ability of the second unlabeled antibody to compete with the first antibody for binding to TIGIT is tested. The second antibody may be present in hybridoma supernatant. As a control, immobilized TIGIT is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to TIGIT, excess unbound antibody is removed and the amount of label associated with immobilized TIGIT is measured. A significant decrease in the amount of label associated with immobilized TIGIT in the test sample compared to the control sample indicates that the second antibody competes with the first antibody for binding to TIGIT. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0182] The present disclosure provides assays for identifying anti-TIGIT antibodies with biological activity. Biological activity may include, for example, activation of immune cells or immune activation reporter genes (e.g., NFAT reporter genes). Antibodies with such biological activity in vivo and / or in vivo are further provided.

[0183] 2.11 Immunoconjugates The presently disclosed subject matter further provides immunoconjugates, which comprise an antibody disclosed herein conjugated to one or more detection probes and / or cytotoxic agents (e.g., chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof)), or radioisotopes. For example, an antibody or antigen-binding portion of the disclosed subject matter can be operably linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or other means) to one or more other binding molecules (e.g., another antibody, antibody fragment, peptide, or binding mimetic).

[0184] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), in which the antibody is conjugated to one or more drugs, and the drugs are maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 106). 235), auristatins such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatin, and calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. [Cancer Research] 58:2925-2928 (1998)) and anthracyclines such as daunomycin or doxorubicin (Kratz et al., Current Med Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358- 362 (2006), Torgov et al., Bioconj. Chem. [Bioconjugate Chemistry] 16:717-721 (2005), Nagy et al., Proc. Natl. Acad. Sci. USA [Publication of the National Academy of Sciences of the United States of America] 97:829-834 (2000), Dubowchik et al., Bioorg. & Med. Chem. Letters [Bioorganic Chemistry and Medicinal Chemistry Communications] 12:1529-1532 (2002), King et al., J Med. Chem. 45:4336-4343 (2002), and U.S. Patent No. 6,630,579), methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, larotaxane, thiotaxane, and otataxane, trichothecenes, and CC1065.

[0185] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-swellin, abrin, dianthin, Phytolaca americana (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, jatrocin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0186] In some examples, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. Multiple radioisotopes may be used in the production of radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may include radioactive atoms used in scintillation studies, such as tc-99m or 1123, or spin labels used in nuclear magnetic resonance (nMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0187] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents (e.g., N-succinimidyl-3-(2-pyridinedimercapto)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfan (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), disazo compounds (e.g., bis(p-azidobenzoyl)hexanediamine), double nitrogen derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., tolylene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene)). For example, ricin immunotoxins may be prepared as described in Vitetta et al., Science, 238:1098 (1987). Carbon-4-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine-pentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker may also be a "cleavable linker" that facilitates release of the cytotoxic drug in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (see Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[0188] Immunoconjugates or ADCs herein expressly cover such conjugates prepared using crosslinkers, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA), which are commercially available.

[0189] 3.How to use The presently disclosed subject matter further provides methods of using the disclosed antibodies (e.g., anti-TIGIT antibodies). In some embodiments, these methods relate to therapeutic uses of the presently disclosed antibodies. In some embodiments, these methods relate to diagnostic uses of the presently disclosed antibodies.

[0190] 3.1 Treatment method The present disclosure provides methods and uses of any of the antibodies disclosed herein (e.g., anti-TIGIT antibodies) for treating diseases and conditions or enhancing immune responses. In some embodiments, antibodies and / or pharmaceutical compositions comprising the antibodies disclosed herein can be administered to a subject (e.g., a mammal (e.g., a human)) to treat diseases and conditions or enhance immune responses. In some embodiments, these diseases and conditions are related to immune checkpoint inhibition and / or aberrant TIGIT activity. In some embodiments, diseases and conditions treatable by the antibodies disclosed herein include, but are not limited to, tumorigenesis (e.g., cancer).

[0191] In some embodiments, the present disclosure provides an anti-TIGIT antibody (or a fragment thereof) described herein for use in the preparation of a medicament. In some embodiments, the present disclosure provides an anti-TIGIT antibody (or a fragment thereof) described herein for use in the preparation of a medicament for the treatment of cancer. In some embodiments, the present disclosure provides an anti-TIGIT antibody (or a fragment thereof) described herein for use in the treatment of cancer in a subject. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an anti-TIGIT antibody (or a fragment thereof) described herein for use in the treatment of cancer in a subject. In some embodiments, the cancer may be a blood cancer (e.g., leukemia, leukemia, and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various cancers (including prostate cancer and small cell lung cancer). Suitable cancers further include any known cancer in the field of oncology, such as astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primary neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma and their liver metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatocarcinoma, cholangiocarcinoma, synovioma, , mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, hidradenoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, medulloblastoma, medullopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia macroglobulinemia), breast tumors (e.g., ductal adenocarcinoma and lobular adenocarcinoma), cervical squamous cell carcinoma and adenocarcinoma, uterine epithelial carcinoma and ovarian epithelial carcinoma, prostate cancer, transitional squamous cell carcinoma of the bladder, B and T lymphomas (nodular and dispersed), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

[0192] In some embodiments, the cancer may be melanoma, NSCLC, head and neck cancer, urothelial cancer, breast cancer (e.g., triple-negative breast cancer, TNBC), gastric cancer, cholangiocarcinoma, classical Hodgkin's lymphoma (cHL), non-Hodgkin's lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer), esophageal cancer, nasopharyngeal carcinoma (NPC), biliary tract cancer, colorectal cancer, cervical cancer, or thyroid cancer.

[0193] In some embodiments, the subject to be treated is a mammal (e.g., a human, a non-primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is suspected of having cancer, is at risk of having cancer, or has been diagnosed with cancer or any other disease with aberrant TIGIT expression or activity.

[0194] Diagnostic methods for many cancers or any other diseases exhibiting abnormal TIGIT activity and the clinical description of these diseases are known in the art. Such methods include, but are not limited to, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH). Further details regarding diagnostic methods for abnormal TIGIT activity or expression are described, for example, in Gupta et al. (2009) Mod Pathol. [Modern Pathology] 22(1):128-133; Lopez-Rios et al. (2013) J Clin Pathol. [Journal of Clinical Pathology] 66(5):381-385; Ellison et al. (2013) J Clin Pathol. [Journal of Clinical Pathology] 66(2):79-89; And and Guha et al. (2013) PLoS ONE [Public Science Library Integrated] 8(6):e67782.

[0195] Administration may be by any suitable route, including, for example, intravenous, intramuscular, or subcutaneous. In some examples, the anti-TIGIT antibodies (or fragments thereof) and / or compositions provided herein can be administered in combination with a second, third, or fourth agent (including, for example, an anti-tumor agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or disorder associated with aberrant TIGIT activity. Such agents include, for example, docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and folinic acid), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (an anti-VEGF antibody), FOLFOX-4, infused fluorouracil, folinic acid and oxaliplatin, alfaltinib, gemcitabine, capecitabine, pemetrexed, tecartinib, everolimus, CpG-ODN, rapamycin, lenalidomide, belofinil, endostatin, lapatinib, PX-866, Imprime PGG, and irinotinib. In some embodiments, the anti-TIGIT antibody (or fragment thereof) is conjugated to another agent.

[0196] In some embodiments, the anti-TIGIT antibodies (or fragments thereof) and / or compositions provided herein are administered in combination with one or more other therapies (e.g., radiation therapy, surgery, chemotherapy, and / or targeted therapy). In some embodiments, the anti-TIGIT antibodies (or fragments thereof) and / or compositions provided herein are administered in combination with radiation therapy. In some embodiments, the anti-TIGIT antibodies (or fragments thereof) and / or compositions provided herein are used in combination with radiation therapy to treat neoplasms or cancers as disclosed herein.

[0197] Depending on the indication to be treated and administration-related factors well known to those skilled in the art, the anti-TIGIT antibodies or fragments thereof provided herein are administered in dosages effective to treat the indication while minimizing toxicity and side effects. For cancer treatment, a typical dosage may be, for example, in the range of 0.001 to 1000 μg, although dosages lower or higher than these exemplary ranges are within the scope of the present invention. Daily dosages may be from about 0.1 μg / kg to about 100 mg / kg of total body weight, from about 0.1 μg / kg to about 100 μg / kg of total body weight, or from about 1 μg / kg to about 100 μg / kg of total body weight. As noted above, therapeutic or prophylactic efficacy can be monitored by periodic evaluation of the treated patient. For repeated administration over several days or more, treatments may be repeated depending on symptoms, until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and are within the scope of the present invention. The desired dosage may be delivered by a single bolus administration of the composition, multiple boluses of the composition, or continuous infusion of the composition.

[0198] A pharmaceutical composition containing an anti-TIGIT antibody or a fragment thereof may be administered once, twice, three times, or four times daily. The composition may also be administered less frequently than daily, for example, six times weekly, five times weekly, four times weekly, three times weekly, twice weekly, once weekly, once every two weeks, once every three weeks, once monthly, once every two months, once every three months, or once every six months. The composition may be administered in a sustained-release formulation, for example, via an implant, which gradually releases the composition for use over a period of time and allows the composition to be administered less frequently, for example, once monthly, once every two to six months, once yearly, or even once daily. A sustained-release device (e.g., pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) may also be administered by injection.

[0199] The antibody (or fragment thereof) may be administered in a single daily dose, or the total daily dose may be administered in divided doses two, three, or four times daily. The composition may also be administered less frequently than daily, for example, six times weekly, five times weekly, four times weekly, three times weekly, twice weekly, once weekly, once every two weeks, once every three weeks, once monthly, once every two months, once every three months, or once every six months. The antibody (or fragment thereof) may also be administered in a sustained-release formulation, for example, in an implant, which gradually releases the composition for use over a period of time and allows the composition to be administered less frequently, for example, once monthly, once every two to six months, once yearly, or even as a single dose. Sustained-release devices (e.g., pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) may be administered by injection or surgical implantation at various locations.

[0200] Cancer treatment may be evaluated by, for example, but not limited to, tumor regression, tumor weight or size reduction, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression and / or activity. Methods for determining therapeutic efficacy can be used, including, for example, measuring response by radiological imaging.

[0201] In some embodiments, the therapeutic effect is measured as percent tumor growth inhibition (%TGI) and is calculated using the equation 100-(T / Cx100), where T is the mean relative tumor volume of treated tumors and C is the mean relative tumor volume of untreated tumors. In some embodiments, the %TGI may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or greater than 95%.

[0202] 3.2 Diagnostic and imaging methods Labeled anti-TIGIT antibodies, fragments thereof, and derivatives or analogs thereof are used diagnostically to detect, diagnose, or monitor diseases and / or disorders associated with TIGIT expression, aberrant expression, and / or activity. For example, anti-TIGIT antibodies (or fragments thereof) provided herein may be used in in situ, in vivo, ex vivo, and in vitro diagnostic or imaging assays. Methods for detecting TIGIT polypeptide expression include (a) measuring polypeptide expression in cells (e.g., tissues) or body fluids of an individual using one or more antibodies of the invention, and (b) comparing the gene expression level with a standard gene expression level, wherein an increase or decrease in the measured gene expression level compared to the standard expression level indicates aberrant expression.

[0203] Another embodiment herein includes methods for diagnosing a disease or disorder associated with TIGIT expression or aberrant expression in an animal (e.g., a mammal, e.g., a human). These methods include detecting a TIGIT molecule in the mammal. In some embodiments, the diagnosis includes (a) administering to the mammal an effective amount of a labeled anti-PD-1 antibody (or fragment thereof); (b) waiting a period of time after administration to allow preferential enrichment of the labeled anti-TIGIT antibody at sites in the subject expressing the TIGIT molecule (and removing unbound labeled molecule to background levels); (c) determining the background level; and (d) detecting the labeled molecule in the subject, with detected labeled molecule levels higher than the background level indicating that the subject has a particular disease or disorder associated with TIGIT expression or aberrant expression. The background level may be determined in different ways, and these methods include comparing the amount of detected labeled molecule to a standard value previously determined for a particular system.

[0204] The anti-TIGIT antibodies (or fragments thereof) herein may be used to measure protein levels in biological samples using classical immunohistology methods well known to those skilled in the art (see, e.g., Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are known in the art and include enzyme labels (e.g., glucose oxidase) and iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, 112 In, 111 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 These include radioisotopes such as Ru, luminol, and fluorescent labels (eg, fluorescein, rhodamine, and biotin).

[0205] Techniques known in the art can be applied to labeling antibodies (or fragments thereof) according to the present disclosure, including, but not limited to, the use of bifunctional conjugates (see, e.g., U.S. Patent Nos. 5,756,065, 5,714,631, 5,696,239, 5,652,361, 5,505,931, 5,489,425, 5,435,990, 5,428,139, 5,342,604, 5,274,119, 4,994,560, and 5,808,003).

[0206] Alternatively or additionally, the level of nucleic acid or mRNA encoding a TIGIT polypeptide in the cells may be measured, for example, using fluorescent in situ hybridization (FISH; see WO98 / 45479 published October 1998) using a nucleic acid-based probe corresponding to a nucleic acid encoding EGFR or its complementary sequence, DNA blotting, RNA blotting, or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). TIGIT overexpression can also be studied by measuring shed antigen in biological fluids (e.g., serum), for example, using antibody-based assays (see, further, e.g., U.S. Pat. No. 4,933,294, published June 12, 1990; WO 91 / 05264, published April 18, 1991; U.S. Pat. No. 5,401,638, published March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above assays, various in vivo and ex vivo assays are available to those skilled in the art. For example, cells within a mammal can be exposed to an antibody, optionally labeled with a detectable label (e.g., a radioisotope), and binding of the antibody to the cells assessed, for example, by radioactive external scanning or by analysis of a sample (e.g., a biopsy or other biological sample) taken from a mammal previously exposed to the antibody.

[0207] 4. Drug Formulations The presently disclosed subject matter further provides pharmaceutical formulations comprising one or more antibodies disclosed herein and a pharmaceutically acceptable carrier agent. In some examples, pharmaceutical compositions may comprise a combination of multiple (e.g., two or more) antibodies and / or antigen-binding portions thereof of the presently disclosed subject matter. In some examples, pharmaceutical compositions of the present disclosure may comprise one or more anti-TIGIT antibodies.

[0208] In some embodiments, the disclosed drug formulations may be prepared by combining an antibody having a desired purity with one or more optional pharmaceutically acceptable carrier agents (see Remington's Pharmaceutical Sciences, 16th ed., edited by Osol, A. (1980)) in the form of a lyophilized formulation or an aqueous solution. For example, lyophilized antibody formulations are described in, but not limited to, U.S. Pat. No. 6,267,958. In some embodiments, aqueous antibody formulations may include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer. In some examples, the antibody may have a purity of greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9%.

[0209] Pharmaceutically acceptable carrier agents are typically nontoxic to recipients at the dosages and concentrations employed, and include buffers (e.g., phosphate, citrate, and other organic acids), antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl parabens (e.g., methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3-pentanol, and meta-cresol), low molecular weight (less than about 10 residues) polypeptides and proteins. Examples of suitable pharmaceutically acceptable carrier agents include, but are not limited to, carbohydrates (e.g., serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins, chelating agents (e.g., EDTA), sugars (e.g., sucrose, mannitol, trehalose, or sorbitol), counterions (e.g., sodium) forming salts, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants (e.g., polyethylene glycol (PEG)). Exemplary pharmaceutically acceptable carrier agents herein include mesenchymal drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX). (登録商標) , Baxter International, Inc. Several exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Nos. 2005 / 0260186 and 2006 / 0104968. In some embodiments, the sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinases).

[0210] The carrier agent may be 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., anti-TIGIT antibody) may be coated in a material to protect the compound from the effects of acids and other natural conditions that may inactivate the compound.

[0211] The pharmaceutical compositions of the present disclosure may be used in combination therapy, i.e., may be administered in combination with other drugs. In some embodiments, the pharmaceutical compositions disclosed herein may further comprise one or more active ingredients, which are essential for the particular indication being treated, e.g., have complementary activities and do not adversely affect each other. In some embodiments, the pharmaceutical formulation may also comprise a second active ingredient for treating the same disease being treated by the first therapeutic agent. Such active ingredients are present in a suitable combination in amounts effective for the desired purpose. For example, but not limited to, the formulations of the present disclosure may further comprise one or more active ingredients, which are essential for the particular indication being treated, preferably have complementary activities and do not adversely affect each other. For example, it may be desirable to further provide a second therapeutic agent for treating the same disease. Such active ingredients are present in a suitable combination in amounts effective for the desired purpose.

[0212] The compositions of the present disclosure may be administered by various methods known in the art. The route and / or mode of administration will depend on the desired results. These active compounds can be prepared using carriers that protect the compound from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can also be used. Many methods for preparing such formulations are described, for example, in "Sustained and Controlled Release Drug Delivery Systems," edited by JR Robinson, Marcel Dekker, Inc., New York, 1978. In some embodiments, the drug compositions are produced under U.S. Food and Drug Administration Good Manufacturing Practice (GMP) conditions.

[0213] Sustained-release formulations containing the disclosed antibodies may also be prepared. Suitable examples of sustained-release formulations include semipermeable matrices containing the antibody in solid hydrophobic polymers, which matrices are in the form of shaped articles (e.g., films or microcapsules). In some embodiments, the active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or coarse emulsions, respectively. Such techniques are described in Remington's Pharmaceutical Sciences, 16th ed., edited by Osol, A. (1980).

[0214] To administer the antibodies of the present disclosure by some routes of administration, it may be necessary to coat the compound with or administer the compound with a material that prevents its inactivation. For example, the compound may be administered to a subject in an appropriate carrier agent (e.g., liposomes) or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

[0215] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The substances used for such media and agents for pharmaceutical activity are well known in the art.

[0216] Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present disclosure is contemplated. Supplementary active compounds may also be doped into the compositions.

[0217] Therapeutic compositions must typically be sterile, essentially isotonic, and stable under production and storage conditions. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable to 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 the like), and suitable mixtures thereof. Proper fluidity can be maintained (for example) by the use of a coating (e.g., lecithin), by the maintenance of a desired 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, polyols (e.g., mannitol, sorbitol), or sodium chloride in the compositions. Prolonged absorption of these injectable compositions can be achieved by including in the composition absorption-delaying agents, such as monostearate and gelatin.

[0218] Sterile injectable solutions may be prepared by incorporating a desired amount of one or more of the disclosed antibodies with an appropriate solvent and a combination of one or more of the ingredients enumerated above, as required, followed by sterilization microfiltration (e.g., filtration through sterile filtration membranes). Typically, dispersions are prepared in the following manner.

[0219] The active compound is incorporated into a sterile vehicle containing a basic dispersion medium and any other desired ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which yields a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution thereof.

[0220] Therapeutic compositions can also be administered using medical devices known in the art.

[0221] For example, therapeutic compositions of the present invention may be administered with a needleless hypodermic injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of implants and modules that can be used in the present disclosure include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate, U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin, U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at precise infusion rates, U.S. Patent No. 4,447,224, which discloses a variable flow rate implantable infusion device for continuous drug delivery, U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with multi-chamber compartments, and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many such implants, delivery systems, and modules are known.

[0222] For therapeutic compositions, the formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of antibody that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of antibody that can be combined with a carrier material to produce a single dosage form will typically be that amount of the composition that produces a therapeutic effect. Typically, this amount will be about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient.

[0223] Dosage forms for topical or transdermal administration of the compositions of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0224] The phrases "parenteral administration" and "administration by parenteral administration" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0225] These pharmaceutical compositions may contain adjuvants, such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of microorganisms may be ensured by sterilization procedures, as described above, and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like, in these compositions. Furthermore, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0226] In some examples, when the antibodies of the present disclosure are administered to humans and animals as drugs, they can be administered alone or in combination with a pharmaceutically acceptable carrier agent as a pharmaceutical composition, which contains, for example, from about 0.01% to about 99.5% (or from about 0.1% to 90%) of an antibody described herein.

[0227] 5.Products The subject matter of the present disclosure further provides articles of manufacture containing materials for use in the treatment, prevention and / or diagnosis of the above-mentioned diseases.

[0228] In some examples, the article of manufacture includes a container and a label or packaging insert on or associated with the container. Non-limiting examples of suitable containers include bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials (e.g., glass or plastic). The container holds a composition effective for treating, preventing, and / or diagnosing a disease (by itself or in combination with another composition) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle).

[0229] In some embodiments, at least one active agent in the composition is an antibody of the presently disclosed subject matter. The label or package insert can indicate that the composition is used for treating a selected condition.

[0230] In some embodiments, the article of manufacture may include (a) a first container containing a composition comprising an antibody of the invention, and (b) a second container containing a composition comprising another cytotoxic or therapeutic agent. In some embodiments, the article of manufacture may further include a packaging insert indicating that the composition can be used to treat a particular condition.

[0231] Alternatively, or additionally, the article of manufacture may further comprise another container, e.g., a second or third container, which may contain a pharmaceutically acceptable buffer, such as, but not limited to, bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0232] [Table A] [Table B] [Table C] [Table D] [Table E] [Table F] [Table G] [Example]

[0233] The following examples are merely illustrative of the subject matter of the present disclosure and should not be construed as limiting in any way.

[0234] example Example 1. Immunization, generation of anti-human TIGIT VHH antibodies, and discovery of the first antibody Recombinant human TIGIT extracellular domain (ECD) protein antigen was purchased from Arco Bio. Llamas were immunized with TIGIT according to known methods. Serum antibody titers were measured by ELISA assay. After three immunizations, high titers (1:100,000) were observed. Whole blood was then collected and PBMCs were isolated. RNA was then isolated from the PBMCs.

[0235] Using a method known in the art, VHH antibody genes were amplified by PCR, purified by DNA agarose gel, and constructed into the phage vector pADL-23c (Antibody Design Labs), which was then transformed into TG1-inducible recipient cells (from Lucigen). The transformed TG1 cells were cultured in Y2T medium. Phages carrying the target VHHs were produced by adding helper phage and co-culturing overnight. Phages in the culture supernatant were collected by centrifugation, and panning of binders against human-TIGIT (h-TIGIT) or cynomolgus monkey-TIGIT (cyno-TIGIT) antigens was performed using streptavidin-conjugated Dynabeads coated with biotinylated h-TIGIT or cyno-TIGIT ECD. After three rounds of panning, h-TIGIT or cyno-TIGIT-bound fragments were eluted and used to infect SS320 cells. Colonies of SS320 cells were selected and cultured in Y2T medium, and IPTG was added to secrete VHH antibodies. Supernatants containing VHH antibodies were screened by ELISA analysis using plates coated with h-TIGIT ECD. Positive h-TIGIT binders were selected and sequenced. 29 clones with different sequences were selected. The binding ability of the VHH antibodies to cyno-TIGIT was further examined by ELISA. The first 21 binders, their CDRs, and VHs are shown in the sequence listing (SEQ ID NO: 1-84).

[0236] Furthermore, we determined the effect of VHH antibody clones on blocking the binding of poliovirus receptor (PVR, also known as CD155) to TIGIT using a blocking ELISA assay, and selected nine clones (clone names: 2B7, 1G1, 1C12, 3G6, 2B10, 3G7, 3F10, 13H11, and 15A5) that exhibited 90% or greater inhibitory activity against the binding of PVR to h-TIGIT.

[0237] Example 2 - Characterization and selection of TIGIT VHH antibodies The antibody clone identified in Example 1 was constructed to prepare a bivalent antibody by adding the human constant heavy chain 2 (CH2) and constant heavy chain 3 (CH3) domains shown in Figure 1C. The constructed bivalent VHH antibody was expressed in ExpiCHO cells, and the protein in the supernatant was collected and purified with Protein A.

[0238] Flow cytometry assays confirmed the binding affinity of bivalent clones to Jurkat cells transfected with human TIGIT. Jurkat cells stably expressing human TIGIT and an NFAT reporter gene were established. Specifically, Jurkat cells were transfected with a human TIGIT expression vector by electroporation, and cells stably expressing human TIGIT were selected using 1 μg / ml puromycin during cell culture. Representative antibody clones were transfected at various concentrations on Jurkat cells (0.2 × 10 ) in 100 μl / well of FACS buffer (PBS containing 1.5% FBS) in a 96-well plate. 6 1 / ml) was incubated with stably expressing h-TIGIT for 30 minutes. After washing, an anti-human IgG Fc secondary antibody (Alexa Fluor 488-coupled) was added. (登録商標)488 AffiniPure goat anti-human IgG, Fcγ fragment specific, Jackson Labs, 1:500 dilution) was added and incubated for 30 minutes. After washing, mean fluorescence intensity was measured by gating on the viable cell population using a CytoFlex (Beckman Coulter) analyzer. Binding affinity was calculated using GraphPad Prism. Representative results are shown in Figures 1A and 1B. Compared to reference Ab1 (the reference anti-human TIGIT antibody disclosed in US 2016 / 0176963 A1 is synthesized according to the disclosed sequence), all of the tested antibodies showed higher binding affinity to cells expressing human TIGIT.

[0239] Jurkat cells stably expressing human TIGIT were transfected with an NFAT reporter gene using electroporation. Cells expressing the NFAT reporter gene were selected with 300 μg / ml hygromycin in the culture medium. Raji cells were stably transfected with human PVR, and transfected cells were selected with 125 μg / ml hygromycin in the culture medium. The effect of antibodies on PVR-mediated inhibition of TCR-induced NFAT reporter gene activity was measured by co-incubating Raji cells expressing human PVR with Jurkat cells expressing human TIGIT and the NFAT reporter gene in the presence of Staphylococcal enterotoxin (SEE, 0.01 ng / ml) for 5 hours. Bright-Glo luciferase assay buffer with substrate (Promega) was added, and luciferase activity was measured by chemiluminescence activity using a plate reader. The ability of antibodies to block TIGIT activity was calculated using nonlinear regression in GraphPad Prims. Representative results are shown in Figure 2. Compared to the reference Ab 1, all tested clones except 3F10 showed similar TIGIT blocking activity.

[0240] Example 3 - Humanization of anti-TIGIT antibodies Two representative clones, 1G1 and 1C12, were selected and their frameworks humanized. Briefly, Igblast analyses were performed using the two clone sequences to search a database of human germline genes. Desired germline sequences were selected, and mutations were performed on the framework sequences to change the framework sequence from llama to human. For the 1C12 clone, human germline IGHV-3-30*10 was used to generate three humanized 1C12 forms (1C12-EREF, 1C12-EREW, and 1C12-GLEW). For the 1G1 clone, human germline IGHV-3-30*01 was used to generate six humanized 1G1 forms (1G1-FG-ERES, 1G1-FA-ERES, 1G1-FA-EREW, 1G1-FA-GLEW, 1G1-FA-GREL, and 1G1-FA-GRES). The construct was cloned into an expression vector, and antibody protein was produced by instantaneous transfection of ExpiCHO and purified by Protein A.

[0241] The binding affinity of the humanized bivalent antibody to human TIGIT was determined by whole cell binding to h-TIGIT stably expressed on Jurkat cells. Briefly, the antibody was added to h-TIGIT-transfected Jurkat cells (100 μl of 0.2 × 10 cells) in FACS buffer. 6 The cells were incubated with anti-human IgG Fc AlexaFluor 488 (1:500) for 30 minutes. As shown in Figure 3A, mean fluorescence intensity was determined using CytoFlex, and nonlinear regression was performed using GraphPad Prism 8.0 to calculate the binding affinity of the antibody to h-TIGIT. Similar binding affinities were observed for all three formats of the 1C12 clone compared to the chimeric parent clone. For the 1G1 clone, the two humanized forms, FG-ERES and FA-ERES, showed similar affinities for h-TIGIT compared to the chimeric clone, whereas two forms (FA-EREW and FA-GLEW) lost binding affinity for h-TIGIT, as shown in Figure 3B.

[0242] Using the previously described method, the activity of humanized antibodies in blocking PVR-mediated inhibition of TCR-mediated NFAT reporter gene activity was determined. Briefly, PVR-transfected Raji cells and Jurkat cells transfected with human TIGIT and an NFAT reporter gene were incubated with various concentrations of antibody for 5 hours in the presence of a low concentration of Staphylococcal enterotoxin (SEE, 0.01 ng / ml). Bright-Glo luciferase assay buffer with substrate (Promega) was added, and luciferase activity was measured. Representative results are shown in Figures 4A and 4B. Compared to the chimeric parent clone and the reference anti-h-TIGIT antibody reference Ab1, all humanized forms of the 1C12 clone had similar efficacy in blocking TIGIT. All humanized forms of 1G1 have similar efficacy to the chimeric parent clone in blocking TIGIT, except for two forms, 1G1-(FA-EREW) and 1G1-(FA-GLEW), which lost their blocking effect.

[0243] Example 4 - Affinity maturation, selection and modification Affinity maturation was performed on the 1G1-FA-ERES clone. Primers were designed to mutate single amino acids in each CDR region. Mutation libraries were prepared using assembly PCR and cloned into phage vectors. Library quality was assessed by DNA sequencing of transformed TG1 cells and clones. Phage generation was performed using helper phage, and phage panning was performed using streptavidin-conjugated Dynabeads coated with biotinylated h-TIGIT ECD or cyno-TIGIT ECD. After two rounds of panning, the panning products were eluted for infection of SS320 cells, and colonies were picked and cultured in Y2T medium with IPTG. VHH antibodies in the supernatants were tested by ELISA. Positive clones against h- and cyno-TIGIT were selected and subjected to whole-cell binding with cells stably expressing h- and cyno-TIGIT. Selected clones were also subjected to PVR-blocking ELISA and detection of the human TIGIT-blocking NFAT reporter gene. EC50 or IC50 values ​​were calculated using GraphPad Prism. The first 25 binders and their CDRs and VHHs are shown in the sequence listing (SEQ ID NOs: 94-177).

[0244] GraphPad Prism was used to plot the EC50 / IC50 correlation in whole cell binding and blocking ELISA assays, and representative data are shown in Figures 5A-5C. 2A3 was identified as having the highest affinity and potency.

[0245] The thermostability of 12 representative clones was tested by heat treatment at temperatures from 25 to 70°C for 60 minutes, and the binding of the treated samples to human TIGIT was detected using ELISA and whole-cell binding flow cytometry, and the results are shown in Figures 6A and 6B. Compared with the other clones, clone 2A3 has higher thermostability.

[0246] The bivalent 2A3 antibody (2A3-Fc) was constructed using human IgG1 CH2 and CH3 domains. The antibody was expressed in ExpiCHO cells and purified using a Protein A column. The 2A3-Fc epitope binding to human TIGIT was investigated using an Octet binding assay, compared to anti-TIGIT reference antibodies Reference Ab1 and Reference Ab2 (reference anti-human TIGIT antibodies with the same amino acid sequence as tiragolumab, synthesized according to the tiragolumab sequence disclosed in US 2017 / 0088613 A1). As shown in Figures 7A-7C, the 2A3 clone bound to a different epitope compared to Reference Ab1 and Reference Ab2.

[0247] The interspecies binding activity of 2A3-Fc to human, cynomolgus monkey, and mouse TIGIT was measured by ELISA assay, and the results are shown in Figures 8A to 8C. 2A3-Fc bound to human and cynomolgus monkey, but not to mouse TIGIT.

[0248] Analysis of the CDR regions of 2A3 identified two hotspots: methionine in CDR2 and aspartic acid and serine in CDR3. The methionine was mutated to leucine and isoleucine, and the aspartic acid was mutated to threonine and glutamic acid. The CDRs and VHHs of the modified antibodies (2A3 ML, 2A3 MI, 2A3 ML_DT (also called 2A3 LT), and 2A3 ML_DE) are shown in the sequence listing (SEQ ID NOs: 178-193). The modified forms were tested in whole-cell binding and NFAT luciferase reporter gene detection, and all of these modified antibodies showed similar properties compared to the parental 2A3 clone. Figure 9 shows representative data for 2A3-LT-Fc (2A3 ML DT), in which M was changed to L and D was changed to T (2A3 ML DT), in which similar human TIGIT binding affinity was observed in whole cell binding assays and similar potency was observed in NFAT reporter assays compared to the parental 2A3-Fc.

[0249] The affinity and potency of 2A3-Fc were compared with the reference anti-TIGIT antibody, reference Ab2. 2A3-Fc exhibited significantly higher affinity than reference Ab2. In whole-cell binding assays, the EC50 value of 2A3-Fc was 0.32 ± 0.06 nM, compared with 0.61 ± 10.13 nM for reference Ab2 (n = 3) (Figure 10A). Compared to reference Ab2, 2A3-Fc also exhibited similar potency in blocking TIGIT in NFAT luciferase reporter gene assays. The EC50 value of 2A3-Fc was 0.48 ± 10.18 nM, compared with 0.72 ± 0.42 nM for reference Ab2 (n = 3) (Figure 10B).

[0250] Example 5 - In vivo efficacy studies The antitumor potential of anti-TIGIT antibodies has been investigated in in vitro experiments. When TIGIT-expressing T cells are contacted with PVR-expressing dendritic cells (DCs) in the tumor microenvironment, PVR expressed on DCs binds to TIGIT on the T cells and inhibits the antitumor activity of T cells, such as the secretion of antitumor cytokines. Treatment with effective anti-TIGIT antibodies blocks the binding of TIGIT to PVR, thereby enhancing the tumor-killing ability of T cells.

[0251] A dendritic cell-T cell mixed lymphocyte reaction (MLR) assay was used to mimic this phenomenon occurring in the tumor microenvironment. CD14-expressing monocytes (monocytes) isolated from PBMCs of healthy donors were cultured in RPMI 1640 medium containing 50 ng / ml GM-CSF and 50 ng / ml IL-4 for 7 days (medium was changed on day 4) to differentiate into DCs. Subsequently, DCs were transferred to RPMI medium containing 100 ng / ml LPS and cultured for 1 day to obtain mature DCs. DCs obtained in this manner stably expressed CD11c (a biomarker for mature DCs), MHC class II (major histocompatibility complex class 2), CD80 (a ligand for CD28), and PVR (a ligand for TIGIT). CD3+ T cells were isolated from PBMCs of other healthy donors using a ThermoFisher T Cell Isolation Kit according to the manufacturer's instructions. Then, 10,000 mature DCs and 200,000 CD3+ T cells were co-cultured in RPMI 1640 medium containing 10% FBS-HI, and different concentrations of anti-TIGIT antibody and control antibody were added to each well. After 48 hours of co-culture at 37°C and 5.0% CO2, IL-2 levels in the supernatant were detected using PerkinElmer's IL-2 AlphaLisa detection kit (AlphaLisa human IL-2 kit: Cat# AL221C). Anti-PD1 antibody served as a positive control, and anti-HER2 antibody served as a negative control.

[0252] As shown in Figure 11, the 2A3-LT-Fc antibody dose-dependently increased the level of IL-2-secreting T cells, which was comparable to that of the positive control anti-PD1 antibody. Conversely, the tiragolumab analog did not increase the level of IL-2-secreting T cells. Because IL-2 is an important anti-tumor cytokine, the anti-TIGIT antibody 2A3-LT-Fc was shown to be able to significantly enhance the anti-tumor activity of T cells in the tumor microenvironment and to exhibit superior anti-tumor activity to the tiragolumab analog.

[0253] Example 6 - Fc Engineering The mechanism by which anti-TIGIT antibodies enhance immune function and antitumor activity is thought to involve not only blocking TIGIT but also binding between antigen-presenting cells (APCs) and effector T cells (CD8+ T cells), binding of the antibody Fc region to APCs via FcγRIIIA, and binding of the VHH domain to TIGIT on effector T cells. To exploit these mechanisms, we engineered two different Fc-enhanced forms: one with DLE mutations (S239D, A330L, and I332E) and one with VPVLL mutations (L235V, F243L, R292P, Y300L, and P396L). Binding of the Fc variants to human FcγRIIIA and FcγRIIB was examined by Octet binding assays of recombinant proteins using the ECDs of FcγRIIIA and FcγRIIB, and the results are shown in Figure 12. Both the DLE and VPVLL mutants have enhanced binding affinity to human FcγRIIIA. The DLE mutant also has enhanced binding affinity to human FcγRIIB, whereas the VPVLL mutant has reduced binding affinity to human FcγRIIB.

[0254] Furthermore, the TIGIT blocking activity of the Fc mutants was examined, where the DLE mutant exhibited reduced TIGIT blocking function, as shown in Figure 13A.

[0255] Furthermore, when an antibody variable region binds to its specific antigen, the Fc region can cross-link FcγRIIIA and induce downstream signaling. Using this mechanism, we examined the effects of Fc mutants on FcγRIIIA-mediated activity using Jurkat cells transfected with human FcγRIIIA and an NFAT reporter gene and 293T cells overexpressing TIGIT. In this assay system, the DLE mutant significantly increased human FcγRIIIA-mediated NFAT reporter gene activity compared to the wild-type Fc format, as shown in Figure 13B.

[0256] [Table 3] [Table 4]

[0257] Example 7 - In vivo efficacy studies The efficacy of 2A3-LT-Fc wt and 2A3-LT-Fc-DLE was evaluated and compared with a reference Ab2 using human TIGIT knock-in C57BL / 6 mice and the MC38 mouse colon cancer model. Mice were inoculated with MC38 tumor cells one week before treatment. The average tumor size was approximately 51 mm. 3 When tumor size reached 1000 mm , treatment was initiated and administered intraperitoneally twice weekly for 2.5 weeks. 2A3-LT-Fc antibody was administered at 6 mg / kg per dose, or reference Ab 2 was administered at 11 mg / kg per dose (same mole / kg as 2A3-LT-Fc antibody). 16 days after treatment, tumor size in mice in several control groups reached the upper limit (2000 mm ). 3 ) was reached. Therefore, 16 days after treatment was the data end point for analysis. In the control group, the mean tumor volume was 1548.76 ± 191 mm after 16 days of treatment. 3 Compared to the control group, treatment with 2A3-LT-Fc wt and 2A3-LT-Fc-DLE significantly reduced tumor growth, with TGI (tumor inhibition) of 38% and 50%, respectively, and RT of 985.05±123 mm. 3 and 802.20±126mm 3 (P values ​​tested by Mann-Whitney test are 0.037 and 0.007, respectively, compared to vehicle control). Treatment with the reference antibody also reduced tumor growth, but this reduction was not statistically significant compared to the control group, with a TGI of 26% and a tumor volume of 1156.16 ± 195 mm. 3 (Figure 14A, Table 5). The results of single tumor volume are shown in Figure 14B. According to these results, both 2A3-LT-Fc and 2A3-LT-Fc-DLE are more effective than the reference Ab 2. In the study, there was no significant change in body weight in each experimental group (Figure 14C), indicating that the treatment was well tolerated.

[0258] [Table 5]

[0259] In addition to the various embodiments shown and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and claimed herein. Thus, specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. The foregoing descriptions of specific embodiments of the disclosed subject matter are provided for purposes of illustration and description. The foregoing descriptions are not intended to be exhaustive or to limit the disclosed subject matter to those disclosed embodiments.

[0260] It will be apparent to those skilled in the art that various modifications and variations can be made in the structure and methodology of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter cover modifications and variations that come within the scope of the appended claims and their equivalents.

[0261] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entireties. The present disclosure provides, for example: [Section 1] 1×10 antibody that binds to TIGIT -7 Antibodies, including single domain antibodies that bind to TIGIT with a KD of M or smaller. [Section 2] The single domain antibody was 1×10 -8 The antibody of item 1, which binds to TIGIT with a KD of M or smaller. [Section 3] The single domain antibody was 5×10 -9 The antibody of item 1 or 2, which binds to TIGIT with a KD of M or smaller. [Section 4] The single domain antibody was 2×10 -9 The antibody according to any one of Items 1 to 3, which binds to TIGIT with a KD of M or smaller. [Section 5] Item 5. The antibody according to any one of Items 1 to 4, wherein the single domain antibody comprises a VHH. [Section 6] Item 6. The antibody according to any one of Items 1 to 5, wherein the single domain antibody or VHH comprises a heavy chain variable region (VH). [Section 7] The single domain antibody cross-competes for binding to TIGIT with a reference anti-TIGIT single domain antibody comprising a heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 96; b) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 98, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 99, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 100; c) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 102, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 103, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 104; d) a heavy chain variable region CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 106, a heavy chain variable region CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 107, and a heavy chain variable region CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 108; e) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 110, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 111, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 112; f) a heavy chain variable region CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 114, a heavy chain variable region CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 115, and a heavy chain variable region CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 116; g) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 118, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 119, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 120; h) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 122, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 123, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 124; i) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 126, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 127, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 128; j) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 130, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 131, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 132; k) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 136; l) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 138, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 139, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 140; m) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 142, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 143, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 144; n) a heavy chain variable region CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 146, a heavy chain variable region CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 147, and a heavy chain variable region CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 148; o) a heavy chain variable region CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 150, a heavy chain variable region CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 151, and a heavy chain variable region CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 152; p) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 154, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 155, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 156; q) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 158, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 159, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 160; r) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 162, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 163, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 164; s) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 166, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 167, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 168; t) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 170, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 171, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 172; u) a heavy chain variable region CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 174, a heavy chain variable region CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 175, and a heavy chain variable region CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 176; v) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 178, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 179, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 180; w) a heavy chain variable region CDR1 comprising an amino acid sequence shown in SEQ ID NO: 182, a heavy chain variable region CDR2 comprising an amino acid sequence shown in SEQ ID NO: 183, and a heavy chain variable region CDR3 comprising an amino acid sequence shown in SEQ ID NO: 184; x) a heavy chain variable region CDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 186, a heavy chain variable region CDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 187, and a heavy chain variable region CDR3 comprising an amino acid sequence as set forth in SEQ ID NO: 188; or y) The antibody according to any one of Aspects 1 to 6, comprising a heavy chain variable region CDR1 comprising amino acids having the sequence shown in SEQ ID NO: 190, a heavy chain variable region CDR2 comprising amino acids having the sequence shown in SEQ ID NO: 191, and a heavy chain variable region CDR3 comprising amino acids having the sequence shown in SEQ ID NO: 192. [Section 8] The single domain antibody comprises a heavy chain variable region, the heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising any one of the amino acid sequences of SEQ ID NOs: 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182, 186, and 190, or a variant thereof having up to about three amino acid substitutions; b) a heavy chain variable region CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, 151, 155, 159, 163, 167, 171, 175, 179, 183, 187, and 191, or a variant thereof having up to about three amino acid substitutions; and c) a heavy chain variable region CDR3 comprising any one of the amino acid sequences of SEQ ID NOs: 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, and 192, or a variant thereof having up to about three amino acid substitutions. [Section 9] The antibody of any one of Items 1 to 8, wherein the single-domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain, and the CDR3 domain comprise the CDR1 domain, the CDR2 domain, and the CDR3 domain, respectively, contained in a reference heavy chain variable region, and the reference heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181, 185, 189, and 193. [Section 10] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence shown in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising amino acids having the sequence shown in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising amino acids having the sequence shown in SEQ ID NO: 96. [Section 11] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence shown in SEQ ID NO: 98, a heavy chain variable region CDR2 comprising amino acids having the sequence shown in SEQ ID NO: 99, and a heavy chain variable region CDR3 comprising amino acids having the sequence shown in SEQ ID NO: 100. [Section 12] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 102, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 103, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 104. [Section 13] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 106, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 107, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 108. [Section 14] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 110, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 111, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 112. [Section 15] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 114, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 115, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 116. [Section 16] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 118, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 119, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 120. [Section 17] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 122, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 123, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 124. [Section 18] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 126, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 127, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 128. [Section 19] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 130, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 131, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 132. [Section 20] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 134, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 135, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 136. [Section 21] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 138, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 139, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 140. [Section 22] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 142, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 143, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 144. [Section 23] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 146, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 147, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 148. [Section 24] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 150, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 151, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 152. [Section 25] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 154, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 155, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 156. [Section 26] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 158, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 159, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 160. [Section 27] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 162, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 163, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 164. [Section 28] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 166, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 167, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 168. [Section 29] The antibody according to any one of Aspects 1 to 9, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 170, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 171, and a heavy chain variable region CDR3 compris...

Claims

1. An antibody that binds to TIGIT, including a single domain antibody that binds to TIGIT, wherein the single domain antibody a) a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 96; b) a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 178, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 179, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 180; c) a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 182, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 183, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 184; d) a heavy chain variable region CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 186, a heavy chain variable region CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 187, and a heavy chain variable region CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 188; or e) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 190, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 191, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

192. ,antibody.

2. The antibody of claim 1, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 94, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 95, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

96.

3. The antibody of claim 1, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 186, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 187, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO:

188.

4. The antibody of claim 1, wherein the single domain antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 181, 185, 189 and 193.

5. The antibody of claim 4, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

97.

6. The antibody of claim 4, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

189.

7. The antibody of any one of claims 1 to 6, wherein the antibody comprises an Fc region.

8. The antibody of claim 7, wherein the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions.

9. The antibody of claim 8 , wherein the Fc region comprises an IgG1 Fc region.

10. The antibody of claim 9, wherein the IgG1 Fc region comprises the mutations L235V, F243L, R292P, Y300L and P396L, or the mutations S239D, A330L and I332E.

11. An immunoconjugate comprising the antibody of any one of claims 1 to 10 linked to a therapeutic agent.

12. The immunoconjugate of claim 11 , wherein the therapeutic agent is a cytotoxin or a radioisotope.

13. A pharmaceutical composition comprising: a) an antibody according to any one of claims 1 to 10 or an immunoconjugate according to claim 11 or 12; and b) a pharmaceutically acceptable carrier.

14. A nucleic acid encoding the antibody of any one of claims 1 to 10.

15. A vector comprising the nucleic acid of claim 14.

16. A host cell comprising the nucleic acid of claim 14 or the vector of claim 15.

17. 17. A method for preparing an antibody according to any one of claims 1 to 10, comprising expressing said antibody in a host cell according to claim 16 and isolating said antibody from said host cell.

18. The antibody according to any one of claims 1 to 10, which is used as a drug.

19. The antibody according to any one of claims 1 to 10, which is used in the treatment of cancer.

20. The pharmaceutical composition according to claim 13, which is used as a drug.

21. The pharmaceutical composition according to claim 13, which is used for the treatment of cancer.

22. 22. The antibody of claim 19 or the pharmaceutical composition of claim 21, wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

23. 17. A kit comprising an antibody according to any one of claims 1 to 10, an immunoconjugate according to claim 11 or 12, a pharmaceutical composition according to claim 13, a nucleic acid according to claim 14, a vector according to claim 15, or a host cell according to claim 16.

24. 24. The kit of claim 23, further comprising a manual for the treatment and / or prevention of a neoplasm.

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