EA042731B1 - PROTEINS CONTAINING A SINGLE-CHAIN VARIABLE FRAGMENT BINDING TO CD3 - Google Patents

Description

cross reference

This application claims the priority of Provisional Application US 62/339685, filed May 20

2016, which is incorporated herein by reference in its entirety.

Sequence listing

This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The mentioned ASCII copy, created on May 19, 2017, is called 47517-704_601_SEQ.TXT and has a size of 66042 bytes.

Inclusion by reference

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and were set forth in the present invention. in its entirety.

Prerequisites for the creation of the invention

CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor (TCR) complex and required for T cell activation. Anti-CD3 antibodies have therapeutic targets, including T cell activation. The present invention provides proteins containing a single chain variable fragment that binds to CD3, including multispecific antibodies containing it.

The essence of the invention

In one embodiment, a protein is disclosed comprising a single chain variable fragment that binds to CD3, wherein said protein comprises a heavy chain variable region (VH), a light chain variable region (VL), and a linker, wherein the VH contains complementarity determining regions HC CDR1, HC CDR2 and HC CDR3, while VL contains the complementarity determining regions LC CDR1, LC CDR2 and LC CDR3, where (a) the amino acid sequence of HC CDR1 is represented in GX ₁ X ₂ X 3 _{NX 4} YX ₅ X ₆ N (SEQ ID NO: 2 ), X ₁ is phenylalanine or asparagine, X2 is threonine, glutamic acid or methionine, X ₃ is phenylalanine or tyrosine, X ₄ is lysine, threonine, glycine, asparagine or glutamic acid, X ₅ is alanine or proline , X ₆ is methionine, leucine, valine or isoleucine; (b) the amino acid sequence of HC CDR2 is shown in RIRSX ₇ X ₈ NX ₉ YX ₁₀ TX ₁₁ YX ₁₂ DX ₁₃ VK (SEQ ID NO: 3), X ₇ is lysine or glycine, X ₈ is tyrosine or serine, X ₉ is asparagine or lysine, X ₁₀ is alanine or glutamic acid, Xc is tyrosine or glutamic acid, X ₁₂ is alanine or lysine, X ₁₃ is serine, glutamic acid, aspartic acid, alanine or glutamine; (c) the amino acid sequence of HC CDR3 is shown in HX ₁₄ NFX ₁₅ X ₁₆ SX ₁₇ ISYWAX ₁₈ (SEQ ID NO: 4), X ₁₄ is glycine, alanine or threonine, X ₁₅ is glycine or asparagine, X ₁₆ is asparagine or aspartic acid, X ₁₇ is tyrosine, histidine, proline, glutamine, leucine or glycine, X ₁₈ is tyrosine or threonine; (d) the amino acid sequence of LC CDR1 is represented in X ₁₉ X ₂₀ X ₂₁ X ₂₂ GX ₂₃ VX ₂₄ X ₂₅ GX ₂₆ YPN (SEQ ID NO: 5), X ₁₉ is glycine or alanine, X ₂₀ is serine or glutamic acid , X ₂₁ is serine or tyrosine, X ₂₂ is threonine, phenylalanine, lysine or serine, X ₂₃ is alanine or tyrosine, X ₂₄ is threonine or valine, X ₂₅ is serine, aspartic acid, lysine, histidine or valine , X20 is asparagine or tyrosine; (e) the amino acid sequence of LC CDR2 is shown in GX ₂₇ X ₂₈ X ₂₉ X ₃₀ X ₃₁ P (SEQ ID NO: 6), X ₂₇ is threonine or isoleucine, X ₂₈ is lysine, glutamic acid, tyrosine, asparagine or serine X ₂₉ is phenylalanine, leucine, glutamic acid, isoleucine, methionine or valine, X30 is leucine, asparagine or glycine, X ₃₁ is alanine or valine; and (f) the amino acid sequence of LC CDR3 is represented in X ₃₂ LWYX ₃₃ NX ₃₄ WX ₃₅ (SEQ ID NO: 7), X ₃₂ is valine, threonine or alanine, X ₃₃ is serine, aspartic acid or alanine, X ₃₄ is is arginine or serine, X ₃₅ is valine, isoleucine or alanine, while ^X 1, X2, ^X 3, ^X 4, X 5, ^X 6, ^X 7, ^X 8, ^X 9, ^X 10, ^X 11 ^{, X} 12,X13, ^X14 , ^X15 , ^X16 ,X17, ^X18 ,X19,X20,X21,X22, ^X23 , ^X24 , ^X25 , _X26 , _X27 , _X28 , _X29 , _X30 , X ₃₁ , X ₃₂ , X ₃₃ , X ₃₄ and X ₃₅ are not simultaneously phenylalanine, threonine, phenylalanine, lysine, alanine, methionine, lysine, tyrosine, asparagine, alanine, tyrosine, alanine, serine, glycine, glycine, asparagine, tyrosine , tyrosine, glycine, serine, serine, threonine, alanine, threonine, serine, asparagine, threonine, lysine, phenylalanine, leucine, alanine, valine, serine, arginine and valine, respectively.

In some embodiments, a CD3-binding single-chain variable fragment-containing protein has the following formula: f1-r1-f2-r2-f3-r3-f4-r4-f5-r5-f6-r6-f7, where r1 is SEQ ID NO: 2; r2 is SEQ ID NO: 3; r3 is SEQ ID NO: 4; r4 is SEQ ID NO:5; r5 is SEQ ID NO:6; and r6 is SEQ ID NO: 7; and where f ₁ , f ₂ , f ₃ , f ₄ and f ₅ represent the remnants of the frame sections, selected so

- 1 042731 in such a way that said protein is at least eighty percent identical to the amino acid sequence shown in SEQ ID NO: 22.

In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises an amino acid sequence wherein rl is SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO: 40. In some embodiments, a protein containing a single-chain variable fragment that binds to CD3 contains an amino acid sequence in which r2 is SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, or SEQ ID NO: 50. where r3 is SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 50, or SEQ ID NO: 60. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r4 is SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73 In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r5 is SEQ ID NO: 74, SEQ ID NO: 75 , SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, or SEQ ID NO: 86. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r6 is SEQ ID NO: 87, SEQ ID NO : 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93. a sequence in which r1 is SEQ ID NO: 39, r2 is SEQ ID NO: 49, r3 is SEQ ID NO: 51, r4 is SEQ ID NO: 61, r5 is SEQ ID NO: 86 and r6 is SEQ ID NO: 87. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 30, r2 is SEQ ID NO: 43, r4 is SEQ ID NO: 64 and r6 is SEQ ID NO: 89. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r3 is SEQ ID NO: 55, r4 is SEQ ID NO: 67, r5 is SEQ ID NO: 77, and r6 is SEQ ID NO: 92. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 31, r2 is SEQ ID NO: 42, r3 is SEQ ID NO: 60, r4 is SEQ ID NO: 64, r5 is SEQ ID NO: 79, and r6 is SEQ ID NO: 91. In some embodiments, implementation of the protein containing a single-chain variable fragment that binds to CD3 contains an amino acid sequence in which r1 is SEQ ID NO: 35, r2 is SEQ ID NO: 46, r3 is SEQ ID NO: 56, r4 is SEQ ID NO: 68 and r5 is SEQ ID NO: 75. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 32, r2 is SEQ ID NO: 47, r3 is SEQ ID NO: 56, r4 is SEQ ID NO: 65, r5 is SEQ ID NO: 80, and r6 is SEQ ID NO: 87. In some embodiments, a protein containing a single chain variable fragment that binds c CD3, contains the amino acid sequence in which r1 is SEQ ID NO: 29, r2 is SEQ ID NO: 44, r3 is SEQ ID NO: 52, r4 is SEQ ID NO: 73 and r5 is SEQ ID NO: 76. In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 33, r2 is SEQ ID NO: 48, r3 is SEQ ID NO: 57, r4 is SEQ ID NO: 69, and r5 is SEQ ID NO: 74. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 38, r4 is SEQ ID NO: 62; and r5 is SEQ ID NO: 81. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 37, r3 is is SEQ ID NO: 53, r4 is SEQ ID NO: 70, r5 is SEQ ID NO: 82, and r6 is SEQ ID NO: 88. In some embodiments, a CD3 binding single chain variable fragment protein

- 2 042731 contains the amino acid sequence in which rl is SEQ ID NO: 34, r2 is SEQ ID NO: 47, r3 is SEQ ID NO: 56, r4 is SEQ ID NO: 68 and r5 is SEQ ID NO: 75. In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 29, r3 is SEQ ID NO: 54, r4 is SEQ ID NO: 71 and r5 is SEQ ID NO: 83. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein rl is SEQ ID NO: 33, r2 is SEQ ID NO: 41, r4 is SEQ ID NO: 63, r5 is SEQ ID NO: 84, and r6 is SEQ ID NO: 90. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is is SEQ ID NO: 30, r2 is SEQ ID NO: 44, r3 is SEQ ID NO: 58, r4 is SEQ ID NO: 66, and r5 is SEQ ID NO: 85. In some embodiments, a protein comprising the CD3-binding single chain variable fragment contains the amino acid sequence where r1 is SEQ ID NO: 40, r2 is SEQ ID NO: 45, r3 is SEQ ID NO: 56, r5 is SEQ ID NO: 78, and r6 is SEQ ID NO: 93. In some embodiments, the CD3 binding single chain variable fragment-containing protein comprises an amino acid sequence wherein r1 is SEQ ID NO: 36, r2 is SEQ ID NO: 50, r3 is is SEQ ID NO: 59, r4 is SEQ ID NO: 72, and r5 is SEQ ID NO: 75.

In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises an amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 94, and SEQ ID NO: 95. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the protein containing a CD3-binding single-chain variable fragment contains the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, embodiments, the CD3-binding single chain variable fragment-containing protein comprises the amino acid sequence shown in SEQ ID NO: 19. In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises the amino acid sequence shown in SEQ ID NO: 94. In some embodiments, the CD3 binding single chain variable fragment containing protein comprises the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the CD3 binding single chain variable fragment containing protein contains the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the CD3-binding single chain variable fragment-containing protein comprises the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises the amino acid the sequence shown in SEQ ID NO: 13. In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the CD3-binding single-chain variable fragment-containing protein. CD3 contains the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the protein containing the CD3-binding single chain variable fragment contains the amino acid sequence shown in SEQ ID NO: 17. In some embodiments, the protein containing the single chain variable fragment the CD3-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 18. In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, the protein , containing a single-chain variable fragment that binds to CD3, contains the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, the protein containing a single-chain variable fragment that binds to CD3 contains the amino acid sequence shown in SEQ ID NO: 25. In In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises an amino acid sequence containing a linker, said linker comprising the amino acid sequence shown in GGGGSGGGGSGGGGS (SEQ ID NO: 1). In some embodiments, a protein containing a CD3-binding single-chain variable fragment binds to CD3 selected from human CD3 and cynomolgus CD3. In some embodiments, a protein containing a CD3-binding single-chain variable fragment binds to human CD3 and cynomolgus monkey CD3 with comparable binding affinity (Kd). In some embodiments, a protein comprising a CD3-binding single-chain variable fragment binds to human CD3 with a human Kd (hKd) ranging from about 1 nM to about 200 nM and to cynomolgus monkey CD3 with a cynomolgus monkey Kd (cKd) ranging from about 1 nM to about 300 nM. In some embodiments, the hKd and cKd values are in the range of about 3 nM to about 5 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nM to about 200 nM , from about 210 nM to about 250 nM, from about 260 nM to about 300 nM.

In some embodiments, a protein comprising a CD3-binding single-chain variable fragment binds to human CD3 with human Kd (hKd), binds to cynomolgus monkey CD3 with cynomolgus monkey Kd (cKd), and the hKd and cKd values are about the same as binding affinity for the CD3 protein that has the sequence shown in anti-CD3 wt (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment binds human CD3 with a human Kd (hKd), cynomolgus monkey CD3 binds with a cynomolgus monkey Kd (cKd), and hKd and cKd values are about 1.5-2 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment binds to human CD3 with human Kd (hKd), cynomolgus monkey CD3 binds to cynomolgus monkey Kd (cKd), and hKd and cKd values are about 3-5 times higher. than the binding affinity for the CD3 protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment binds to human CD3 with human Kd (hKd), binds to cynomolgus monkey CD3 with cynomolgus monkey Kd (cKd), and hKd and cKd values are about 6-15 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein comprising a CD3-binding single-chain variable fragment binds to human CD3 with human Kd (hKd), binds to cynomolgus monkey CD3 with cynomolgus monkey Kd (cKd), and hKd and cKd values are about 20-50 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22).

In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 8, and the hKd and cKd values are approximately the same as the binding affinity for CD3 of a protein that has the sequence, presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 8 and hKd and cKd values are in the range of about 3 nM to about 5 nM. In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 9, and the hKd and cKd values are about the same as the binding affinity for CD3 of a protein that has the sequence, presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 9 and hKd and cKd values range from about nM to about 5 nM. In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 10, and the hKd and cKd values are about the same as the binding affinity for CD3 of a protein that has the sequence, presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 10 and hKd and cKd values are in the range of about 3 nM to about 5 nM. In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 11, and wherein the hKd and cKd values are about the same as the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 11 and hKd and cKd values are in the range of about 3 nM to about 5 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has an amino acid

- 4 042731 sequence shown in SEQ ID NO: 12, and the hKd and cKd values are about 1.5-2 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 12 and hKd and cKd values are in the range of about 6 nM to about 10 nM. In some embodiments, a protein comprising a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 13 and hKd and cKd values are about 1.5-2 times higher than the binding affinity for the CD3 protein , which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 13 and hKd and cKd values are in the range of about 6 nM to about 10 nM. In some embodiments, a protein containing a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 14 and hKd and cKd values are about 3-5 times higher than the binding affinity for the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 14 and hKd and cKd values are in the range of about 11 nM to 20 nM. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 15 and hKd and cKd values are about 6-15 times higher than the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 15 and hKd and cKd values are in the range of about 25 nM to about 40 nM. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 16 and hKd and cKd values are about 3-5 times higher than the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 16 and hKd and cKd values are in the range of about 11 nM to 20 nM. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 17 and hKd and cKd values are about 6-15 times higher than the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 17 and hKd and cKd values are in the range of about 40 nM to about 60 nM. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 18 and hKd and cKd values are about 3-5 times higher than the binding affinity for CD3 of a protein that contains the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 18 and hKd and cKd values are in the range of about 11 nM to 20 nM. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 19 and hKd and cKd values are about 6-15 times higher than the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 19 and hKd and cKd values are in the range of about 40 nM to 60 nM. In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 20 and hKd and cKd values are about 3-5 times the binding affinity for CD3 of a protein that has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 20 and hKd and cKd values are in the range of about 11 nM to about 20 nM. In some embodiments, a protein comprising a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 21 and hKd and cKd values are about 20-50 times higher than the binding affinity for CD3 of a protein that contains the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments

- 5 042731 protein containing a single-chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 21, and the values of hKd and cKd are in the range from about 125 nm to about 140 nm. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 94 and hKd and cKd values are about 20-50 times higher than the binding affinity for CD3 of a protein that contains the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment protein has the amino acid sequence shown in SEQ ID NO: 94 and hKd and cKd values are in the range of about 100 nM to about 120 nM. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 95 and hKd and cKd values are about 20-50 times higher than the binding affinity for CD3 of a protein that contains the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3 binding single chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 95 and hKd and cKd values are in the range of about 100 nM to about 120 nM. In another embodiment, the present invention provides a protein containing a single chain variable fragment that binds to CD3, containing the sequence shown in SEQ ID NO: 22 (wt anti-CD3), wherein one or more amino acid residues selected from amino acid positions 27, 28, 29, 31, 33, 34, 54, 55, 57, 59, 61, 63, 65, 102, 105, 106, 108, 114, 163, 164, 165, 166, 168, 170, 171, 173, 193, 194, 195, 196, 197, 231, 235, 237 and 239 are substituted, amino acid position 27 is replaced by asparagine, amino acid position 28 is substituted by glutamic acid or methionine, amino acid position 29 is substituted by tyrosine, amino acid position 31 is substituted by asparagine, glycine, glutamic acid or threonine, amino acid position 33 substituted by proline, amino acid position 34 substituted by valine, leucine or isoleucine, amino acid position 54 substituted by glycine, amino acid position 55 substituted by serine, amino acid position 57 substituted by lysine, amino acid position 59 substituted by glutamic acid, amino acid position 61 is substituted with glutamic acid, amino acid position 63 is substituted with lysine, amino acid position 65 is substituted with aspartic acid, glutamic acid, alanine or glutamine, amino acid position 102 is substituted with alanine or threonine, amino acid position 105 is substituted with asparagine, amino acid position 106 is substituted with aspartic acid, amino acid position 108 is substituted histidine, proline, glutamine, glycine or leucine, amino acid position 114 substituted with threonine, amino acid position 163 substituted with alanine, amino acid position 164 substituted with glutamic acid, amino acid position 165 substituted with tyrosine, amino acid position 166 substituted with phenylalanine, lysine or serine, amino acid position 168 substituted with tyrosine , amino acid position 170 is substituted with valine, amino acid position 171 is substituted with aspartic acid, lysine, valine or histidine, amino acid position 173 is substituted with tyrosine, amino acid position 193 is substituted with isoleucine, amino acid position 194 is substituted with glutamic acid, tyrosine, asparagine or serine, amino acid position 195 is substituted with leucine , glutamic acid, isoleucine, methionine, or valine, amino acid position 196 is substituted with asparagine or glycine, amino acid position 197 is substituted with valine, amino acid position 231 is substituted with threonine or alanine, amino acid position 235 is substituted with aspartic acid or alanine, amino acid position 237 is substituted with serine, and amino acid position 239 substituted by alanine or isoleucine. In some embodiments, a protein containing a CD3-binding single-chain variable fragment contains one or more additional substitutions at amino acid positions other than positions 27, 28, 29, 31, 33, 34, 54, 55, 57, 59, 61, 63, 65, 102, 105, 106, 108, 114, 163, 164, 165, 166, 168, 170, 171, 173, 193, 194, 195, 196, 197, 231, 235, 237, and 239. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 27. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 28. In some embodiments, the single-chain variable fragment-containing protein The CD3-binding protein contains a substitution at position 29. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 31. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 33. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 34. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 54. In some embodiments, the protein , containing a single-chain variable fragment that binds to CD3, contains a substitution at position 55. In some embodiments, a protein containing a single-chain variable fragment,

- 6 042731 that binds to CD3 contains a substitution at position 57. In some embodiments, the protein containing the single-chain variable fragment that binds to CD3 contains a substitution at position 59. In some embodiments, the protein that contains the single-chain variable fragment that binds to CD3 contains a substitution at position 61. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 63. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 65. In some embodiments, In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 102. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 105. The CD3-binding protein contains a substitution at position 106. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 108. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 114. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 163. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 164. In some embodiments, the protein containing a CD3-binding single-chain variable fragment contains a substitution at position 165. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a substitution at position 166. In some embodiments, a CD3-binding single-chain variable fragment-containing protein , contains a substitution at position 168. In some embodiments, the protein containing the CD3-binding single-chain variable fragment contains a substitution at position 170. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 171. B In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 173. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 193. the CD3-binding fragment contains a substitution at position 194. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 195. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 196. In some embodiments, the CD3-binding single chain variable fragment-containing protein contains a substitution at position 197. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 231. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 235. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a substitution at position 237. In some embodiments, the CD3-binding single-chain variable fragment-containing protein with CD3, contains a substitution at position 239. In some embodiments, a protein containing a single-chain variable fragment that binds to CD3 contains substitutions at positions 34, 65, 102, 163, 197, and 231. In some embodiments, a protein containing a single-chain variable fragment CD3-binding contains substitutions at positions 28, 57, 166, and 235. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 108, 168, 194, and 239. In some embodiments, the protein containing a CD3-binding single-chain variable fragment contains substitutions at positions 28, 55, 114, 166, 195, and 237. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 31, 63, 108, 170 and 194. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 29, 65, 108, 166, 195, and 231. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 27, 59, 102, 173, and 194. In some embodiments, a protein comprising a CD3-binding single chain variable fragment contains substitutions at positions 31, 65, 108, 171, and 193. In some embodiments, a protein comprising the CD3-binding single-chain variable fragment contains substitutions at positions 34, 164, and 195. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 33, 105, 171, 195, and 231. In some embodiments, implementation of a protein containing a single-chain variable fragment that binds to CD3 contains substitutions at positions 31, 65, 108, 170 and 194. In some

In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 27, 106, 171, and 195. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 31, 54 , 165, 196, and 235. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 28, 59, 108, 166, and 196. In some embodiments, the CD3-binding single-chain variable fragment-containing protein , contains substitutions at positions 34, 61, 108, 194, and 239. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains substitutions at positions 31, 65, 108, 171, and 194.

In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 34, 65, 102, 163, 197, and 231 are substituted, and wherein the hKd and cKd values are about the same as the affinity binding to a CD3 protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 34, 65, 102, 163, 197, and 231 are substituted, and wherein hKd and cKd values are in the range of about 3 nM up to about 5 nM. In some embodiments, a protein containing a CD3-binding single-chain variable fragment contains a sequence in which amino acid positions 28, 57, 166, and 235 are substituted, and wherein the hKd and cKd values are the same as the binding affinity for CD3 a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 28, 57, 166, and 235 are substituted and hKd and cKd values are in the range of about 3 nM to about 5 nM .

In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 108, 168, 194, and 239 are substituted, and wherein the hKd and cKd values are about the same as the binding affinity for CD3 protein which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 108, 168, 194, and 239 are substituted and hKd and cKd values are in the range of about 3 nM to about 5 nM . In some embodiments, a protein containing a CD3-binding single-chain variable fragment contains a sequence in which amino acid positions 28, 55, 114, 166, 195, and 237 are substituted, and wherein the hKd and cKd values are about the same as the affinity binding to a CD3 protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 28, 55, 114, 166, 195, and 237 are substituted, and wherein hKd and cKd values are in the range of about 3 nM up to about 5 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 63, 108, 170, and 194 are substituted and hKd and cKd values are about 1.5-2 times higher than the binding affinity for the CD3 protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 63, 108, 170, and 194 are substituted, and wherein the hKd and cKd values are in the range of about 6 nM to about 10 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 29, 65, 108, 166, 195, and 231 are substituted, and wherein hKd and cKd values are about 1.5-2 - times higher than the binding affinity for CD3 protein, which has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 29, 65, 108, 166, 195, and 231 are substituted, and wherein hKd and cKd values are in the range of about 6 nM up to about 10 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 27, 59, 102, 173, and 194 are substituted, and wherein the hKd and cKd values are about 3-5 times higher than the binding affinity for the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 27, 59, 102, 173, and 194 are substituted and hKd and cKd values are in the range of about 11 nM to 20 nM. In some variants

- 8 042731 implementation of the protein containing a single-chain variable fragment that binds to CD3 contains a sequence in which amino acid positions 31, 65, 108, 171 and 193 are substituted, and while the values of hKd and cKd are approximately 6-12 times higher than the binding affinity for the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 65, 108, 171, and 193 are substituted, and wherein hKd and cKd values are in the range of about 25 nM to about 40 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 34, 164, and 195 are substituted, and wherein the hKd and cKd values are about 3-5 times higher than the binding affinity for to a CD3 protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 34, 164, and 195 are substituted, with hKd and cKd values ranging from about 11 nM to 20 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 33, 105, 171, 195, and 231 are substituted, and wherein the hKd and cKd values are about 6-15 times higher than binding affinity for the CD3 protein that has the sequence shown in anti-CD3 wt (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 33, 105, 171, 195, and 231 are substituted, and wherein hKd and cKd values are in the range of about 40 nM to about 60 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 65, 108, 170, and 194 are substituted, and wherein the hKd and cKd values are about 3-5 times higher than binding affinity for the CD3 protein that has the sequence shown in anti-CD3 wt (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 65, 108, 170, and 194 are substituted and hKd and cKd values are in the range of about 11 nM to 20 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 27, 106, 171, and 195 are substituted, and wherein hKd and cKd values are about 6-15 times higher than the binding affinity in relation to the CD3 protein, which has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 27, 106, 171, and 195 are substituted, with hKd and cKd values in the range of about 40 nM to 60 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 54, 165, 196, and 235 are substituted, and wherein the hKd and cKd values are about 3-5 times higher than binding affinity for the CD3 protein that has the sequence shown in anti-CD3 wt (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 54, 165, 196, and 235 are substituted (10B2) and wherein hKd and cKd values range from about 11 nM to about 20 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 28, 59, 108, 166, and 196 are substituted, and wherein the hKd and cKd values are about 25-50 times higher than the binding affinity for the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 28, 59, 108, 166, and 196 are substituted, and wherein hKd and cKd values are in the range of about 125 nM to about 140 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 34, 61, 108, 194, and 239 are substituted, and wherein the hKd and cKd values are about 20-50 times higher than binding affinity for the CD3 protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 34, 61, 108, 194, and 239 are substituted, and wherein hKd and cKd values are in the range of about 100 nM to about 120 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 65, 108, 171, and 194 are substituted, and when

- 9 042731 this hKd and cKd values are approximately 20-50 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid positions 31, 65, 108, 171, and 194 are substituted, and wherein hKd and cKd values are in the range of about 100 nM to about 120 nM.

In another embodiment, the present invention provides a protein comprising a CD3 binding single chain variable fragment, said protein having the amino acid sequence shown in anti-CD3 wt (SEQ ID NO: 22) comprising a heavy chain variable region (VH), light chain variable region (VL), a linker containing the amino acid sequence shown in GGGGSGGGGSGGGGS (SEQ ID NO: 1), while VH contains complementarity-determining regions CDR1, CDR2 and CDR3, while VL contains complementarity-determining regions LC CDR1, LC CDR2 and LC CDR3 containing at least one mutation in CDR1, CDR2, or CDR3 VH, and LC CDR1, LC CDR2, or LC CDR3 VL, with at least one mutation not located at amino acid positions 26, 30, 32, 35, 50 , 51, 52, 53, 56, 58, 60, 62, 64, ,66, 67, 101, 103, 104, 107, 109, 110, 111, 112, 113, 167, 169, 172, 174, 175, 176, 192, 198, 232, 233, 234, 236, or 238. In some embodiments, the CD3 binding single chain variable fragment-containing protein contains at least one mutation at an amino acid position selected from 27, 28, 29, 31 , 33, 34, 54, 55, 57, 59, 61, 63, 65, 102, 105, 106, 108, 114, 163, 164, 165, 166, 168, 170, 171, 173, 193, 194, 195 , 196, 197, 231, 235, 237, and 239. In some embodiments, amino acid position 34 is mutated to isoleucine, position 65 is mutated to glutamine, position 102 is mutated to alanine, position 163 is mutated to alanine, position 197 is mutated to valine and position 231 mutated to threonine. In some embodiments, amino acid position 28 is mutated to glutamic acid, position 57 is mutated to lysine, position 166 is mutated to phenylalanine, and position 235 is mutated to aspartic acid. In some embodiments, amino acid position 108 is mutated to histidine, position 168 is mutated to tyrosine, position 194 is mutated to serine, and position 239 is mutated to isoleucine. In some embodiments, amino acid position 28 is mutated to methionine, position 55 is mutated to serine, position 114 is mutated to threonine, position 166 is mutated to phenylalanine, position 195 is mutated to leucine, and position 237 is mutated to serine. In some embodiments, amino acid position 31 is mutated to threonine, position 63 is mutated to lysine, position 108 is mutated to proline, position 170 is mutated to valine, and position 194 is mutated to glutamic acid. In some embodiments, amino acid position 29 is mutated to tyrosine, position 65 is mutated to glutamic acid, position 108 is mutated to proline, position 166 is mutated to lysine, position 195 is mutated to glutamic acid, and position 231 is mutated to threonine. In some embodiments, amino acid position 27 is mutated to asparagine, position 59 is mutated to glutamic acid, position 102 is mutated to threonine, position 173 is mutated to tyrosine, and position 194 is mutated to tyrosine. In some embodiments, amino acid position 31 is mutated to asparagine, position 65 is mutated to alanine, position 108 is mutated to glutamine, position 171 is mutated to aspartic acid, and position 193 is mutated to isoleucine. In some embodiments, amino acid position 34 is mutated to valine, position 164 is mutated to glutamic acid, and position 195 is mutated to isoleucine. In some embodiments, amino acid position 33 is mutated to proline, position 105 is mutated to asparagine, position 171 is mutated to lysine, position 195 is mutated to methionine, and position 231 is mutated to alanine. In some embodiments, amino acid position 31 is mutated to glycine, position 65 is mutated to glutamic acid, position 108 is mutated to proline, position 170 is mutated to valine, and position 194 is mutated to glutamic acid. In some embodiments, amino acid position 27 is mutated to asparagine, position 106 is mutated to aspartic acid, position 171 is mutated to histidine, and position 195 is mutated to valine. In some embodiments, amino acid position 31 is mutated to asparagine, position 54 is mutated to glycine, position 165 is mutated to tyrosine, position 196 is mutated to asparagine, and position 235 is mutated to alanine. In some embodiments, amino acid position 28 is mutated to glutamic acid, position 59 is mutated to glutamic acid, position 108 is mutated to leucine, position 166 is mutated to serine, and position 196 is mutated to glycine. In some embodiments, amino acid position 34 is substituted with leucine, amino acid position 61 is substituted with glutamic acid, amino acid position

- 10 042731

108 is substituted with proline, amino acid position 194 is substituted with asparagine, and amino acid position 239 is substituted with alanine. In some embodiments, amino acid position 31 is substituted with glutamic acid, amino acid position 65 is substituted with aspartic acid, amino acid position 108 is substituted with glycine, amino acid position 171 is substituted with valine, amino acid position 194 is substituted with glutamic acid.

In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 34 is mutated to isoleucine, position 65 is mutated to glutamine, position 102 is mutated to alanine, position 163 is mutated to alanine, position 197 is mutated to valine and position 231 is mutated to threonine, and the hKd and cKd values are about the same as the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22 ). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 34 is mutated to isoleucine, position 65 is mutated to glutamine, position 102 is mutated to alanine, position 163 is mutated to alanine, position 197 is mutated to valine and position 231 is mutated to threonine, with hKd and cKd values ranging from about 3 nM to 5 nM. In some embodiments, a protein containing a CD3-binding single-chain variable fragment contains a sequence in which amino acid position 28 is mutated to glutamic acid, position 57 is mutated to lysine, position 166 is mutated to phenylalanine, and position 235 is mutated to aspartic acid. , and while the values of hKd and cKd are about the same as the binding affinity for CD3 protein, which has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment contains a sequence in which amino acid position 28 is mutated to glutamic acid, position 57 is mutated to lysine, position 166 is mutated to phenylalanine, and position 235 is mutated to aspartic acid. , with hKd and cKd values ranging from about 3 nM to about 5 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 108 is mutated to histidine, position 168 is mutated to tyrosine, position 194 is mutated to serine, and position 239 is mutated to isoleucine, and while the values of hKd and cKd are approximately the same as the binding affinity for CD3 protein, which has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 108 is mutated to histidine, position 168 is mutated to tyrosine, position 194 is mutated to serine, and position 239 is mutated to isoleucine, with In this case, the hKd and cKd values range from about 3 nM to about 5 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 28 is mutated to methionine, position 55 is mutated to serine, position 114 is mutated to threonine, position 166 is mutated to phenylalanine, position 195 is mutated to leucine and position 237 is mutated to serine, and the hKd and cKd values are about the same as the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22 ). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 28 is mutated to methionine, position 55 is mutated to serine, position 114 is mutated to threonine, position 166 is mutated to phenylalanine, position 195 is mutated to leucine and position 237 is mutated to serine, with hKd and cKd values ranging from about 3 nM to about 5 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to threonine, position 63 is mutated to lysine, position 108 is mutated to proline, position 170 is mutated to valine, and position 194 is mutated to glutamic acid, with hKd and cKd values about 1.5-2 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22) . In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to threonine, position 63 is mutated to lysine, position 108 is mutated to proline, position 170 is mutated to valine, and position 194 is mutated to glutamic acid with hKd and cKd values ranging from about 6 nM to about 10 nM. In some embodiments, a protein containing a single chain variable

- 11 042731 fragment binding to CD3 contains a sequence in which amino acid position 29 is mutated to tyrosine, position 65 is mutated to glutamic acid, position 108 is mutated to proline, position 166 is mutated to lysine, position 195 is mutated to glutamic acid acid and position 231 are mutated to threonine, with hKd and cKd values about 1.5-2 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 29 is mutated to tyrosine, position 65 is mutated to glutamic acid, position 108 is mutated to proline, position 166 is mutated to lysine, position 195 is mutated to glutamic acid; and position 231 is mutated to threonine, with hKd and cKd values ranging from about 6 nM to about 10 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 27 is mutated to asparagine, position 59 is mutated to glutamic acid, position 102 is mutated to threonine, position 173 is mutated to tyrosine, and position 194 is mutated to tyrosine, with hKd and cKd values about 3-5 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 27 is mutated to asparagine, position 59 is mutated to glutamic acid, position 102 is mutated to threonine, position 173 is mutated to tyrosine, and position 194 is mutated to tyrosine with hKd and cKd values in the range of about 11 nM to 20 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to asparagine, position 65 is mutated to alanine, position 108 is mutated to glutamine, position 171 is mutated to aspartic acid, and position 193 is mutated to isoleucine, with hKd and cKd values about 6-15 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to asparagine, position 65 is mutated to alanine, position 108 is mutated to glutamine, position 171 is mutated to aspartic acid, and position 193 is mutated to isoleucine, with hKd and cKd values ranging from about 25 nM to about 40 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 34 is mutated to valine, position 164 is mutated to glutamic acid, and position 195 is mutated to isoleucine, and where the hKd and cKd values are approximately 3-5 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid position 34 is mutated to valine, position 164 is mutated to glutamic acid, and position 195 is mutated to isoleucine, wherein the hKd and cKd values are in the range from about 11 nM to 20 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 33 is mutated to proline, position 90 is mutated to asparagine, position 105 is mutated to asparagine, position 171 is mutated to lysine, position 195 is mutated to methionine and position 231 is mutated to alanine, with hKd and cKd values about 6-15 times higher than the binding affinity for CD3 of a protein that has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 33 is mutated to proline, position 90 is mutated to asparagine, position 105 is mutated to asparagine, position 171 is mutated to lysine, position 195 is mutated to methionine and position 231 is mutated to alanine, with hKd and cKd values ranging from about 40 nM to 60 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to glycine, position 65 is mutated to glutamic acid, position 108 is mutated to proline, position 170 is mutated to valine, and position 194 is mutated to glutamic acid, with hKd and cKd values about 3-5 times higher than the binding affinity for CD3 protein, which has the sequence shown in anti-CD3 wt (SEQ ID NO: 22). In some

In other embodiments, the CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to glycine, position 65 is mutated to glutamic acid, position 108 is mutated to proline, position 170 is mutated to valine and position 194 are mutated to glutamic acid, with hKd and cKd values ranging from about 11 nM to 20 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 27 is mutated to asparagine, position 106 is mutated to aspartic acid, position 171 is mutated to histidine, and position 195 is mutated to valine, and while the values of hKd and cKd about 6-15 times higher than the binding affinity for CD3 protein, which has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 27 is mutated to asparagine, position 106 is mutated to aspartic acid, position 171 is mutated to histidine, and position 195 is mutated to valine, while the values of hKd and cKd are in the range from about 40 nm to 60 nm. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to asparagine, position 54 is mutated to glycine, position 165 is mutated to tyrosine, position 196 is mutated to asparagine, and position 235 is mutated to alanine, with hKd and cKd values about 3-5 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 31 is mutated to asparagine, position 54 is mutated to glycine, position 165 is mutated to tyrosine, position 196 is mutated to asparagine, and position 235 is mutated to alanine with hKd and cKd values ranging from about 11 nM to about 20 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 28 is mutated to glutamic acid, position 59 is mutated to glutamic acid, position 108 is mutated to leucine, position 166 is mutated to serine and position 196 is mutated to glycine, and the hKd and cKd values are about 20-50 times higher than the binding affinities for the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 28 is mutated to glutamic acid, position 59 is mutated to glutamic acid, position 108 is mutated to leucine, position 166 is mutated to serine and position 196 is mutated to glycine, with hKd and cKd values in the range of about 125 nM to about 140 nM. In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 34 is mutated to leucine, amino acid position 61 is mutated to glutamic acid, amino acid position 108 is mutated to proline, amino acid position 194 is mutated to asparagine, amino acid position 239 is mutated to alanine, and the hKd and cKd values are about 20-50 times higher than the binding affinities for the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein comprises a sequence in which amino acid position 34 is mutated to leucine, amino acid position 61 is mutated to glutamic acid, amino acid position 108 is mutated to proline, amino acid position 194 is mutated to asparagine, amino acid position 239 is mutated to alanine, with hKd and cKd values ranging from about 100 nM to about 120 nM. In some embodiments, a protein comprising a CD3-binding single-chain variable fragment contains a sequence in which amino acid position 31 is mutated to glutamic acid, amino acid position 65 is mutated to aspartic acid, amino acid position 108 is mutated to glycine, amino acid position 171 is mutated mutated to valine and amino acid position 194 is mutated to glutamic acid, and the hKd and cKd values are approximately 20-50 times higher than the binding affinity for CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a CD3-binding single-chain variable fragment-containing protein contains a sequence in which amino acid position 31 is mutated to glutamic acid, amino acid position 65 is mutated to aspartic acid, amino acid position 108 is mutated to glycine, amino acid position 13 042731 position 171 is mutated to valine; and amino acid position 194 is mutated to glutamic acid, with hKd and cKd values ranging from about 100 nM to about 120 nM.

In some embodiments, a protein containing a single chain variable fragment that binds to CD3 does not bind to murine CD3.

In one embodiment, a polynucleotide is provided encoding a protein comprising a single chain variable fragment that binds to CD3, in accordance with the present invention. In one embodiment, a vector is provided that contains the polynucleotide described in the present invention. In one embodiment, a host cell transformed with the vector described in the present invention is provided. In a further embodiment, a pharmaceutical composition is provided comprising (i) a CD3 binding single chain variable fragment-containing protein according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention, or a host cell according to the present invention, and (ii) a pharmaceutically acceptable carrier.

In another embodiment, a method is provided for producing a protein containing a single chain variable fragment that binds to CD3, in accordance with the present invention, this method includes culturing a host transformed or transfected with a vector containing a nucleic acid sequence encoding a protein containing a single chain variable fragment, binding to CD3, in accordance with the present invention, under conditions that allow the expression of a protein containing a single-chain variable fragment that binds to CD3, and isolation and purification of the resulting protein from culture.

In another embodiment, a method is provided for treating or attenuating a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft versus host disease, or host versus graft disease, comprising administering a protein, containing a single-chain variable fragment that binds to CD3, in accordance with the present invention, the subject in need of it. In some embodiments, the subject is a human. In some embodiments, the method further comprises administering the agent in combination with a CD3-binding single-chain variable fragment-containing protein of the present invention.

The present invention, in one embodiment, provides a multispecific binding protein comprising a CD3-binding single-chain variable fragment-containing protein according to the present invention. In one embodiment, an antibody is described comprising a CD3-binding single-chain variable fragment-containing protein according to the present invention. In another embodiment, a multispecific antibody, bispecific antibody, single domain antibody, heavy chain variable domain, peptide or ligand comprising a CD3 binding single chain variable fragment protein according to the present invention is provided. In one embodiment, the present invention describes an antibody comprising a CD3-binding single chain variable fragment-containing protein according to the present invention, said antibody being a scFv antibody. In another embodiment, a multispecific binding protein or antibody is described comprising a CD3-binding single-chain variable fragment-containing protein of the present invention and a serum albumin binding domain.

Brief description of the drawings

The novel features of the invention are detailed in the appended claims. A more complete understanding of the features and advantages of the present invention will be obtained with reference to the following detailed description, which sets forth illustrative embodiments that use the principles of the invention, and the accompanying drawings.

In FIG. 1 shows the titration of a bacteriophage for biotin-CD3ε and biotin-HSA.

In FIG. 2 shows the amino acid sequence of a human CD3 binding protein (SEQ ID NO: 22). HC CDR1 is listed in the first shaded sequence (SEQ ID NO: 23)), HC CDR2 is listed in the second shaded sequence (RIRSKYNNYATYYADSVK (SEQ ID NO: 24)), HC CDR3 is listed in the third shaded sequence (HGNFGNSYISYWAY (SEQ ID NO: 25) ), LC CDR1 is listed in the fourth shaded sequence (GSSTGAVTSGNYPN (SEQ ID NO: 26)), LC CDR2 is listed in the fifth shaded sequence (GTKFLAP (SEQ ID NO: 27)), and CDR3 is listed in the sixth shaded sequence (VLWYSNRWV (SEQ ID NO: 28)).

In FIG. 3 shows the profiles of sixteen clones selected for more accurate Kd determinations.

In FIG. 4 shows the hydrophobic exposure temperature (T _h °C) for several antihuCD3ε scFv variants.

- 14 042731

Detailed description of the invention

Although the present invention shows and describes the preferred embodiments of the present invention, it will be obvious to those skilled in the art that such embodiments are given by way of example only. Without going beyond the scope of this invention, a person skilled in the art can make numerous variations, changes and substitutions. It should be borne in mind that various alternatives to the embodiments of the invention described in the present invention can be used in the practice of this invention. It is intended that the appended claims define the scope of this invention, and that the methods and structures within the scope of that claim, and their equivalents, are embraced by this claim. Some Definitions The terminology used in the present invention is for the purpose of describing particular cases only and does not limit the invention. In the present invention, the indefinite and definite forms of the singular also include plural forms, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms including, includes, having, has, with, or variations thereof are used in the detailed description and/or claims, such terms are intended to be inclusive like the term comprising.

The term approximately or approximately means being within the range of acceptable error for a particular quantity as determined by one skilled in the art, which will depend in part on how the quantity is measured or determined, ie the limitations of the measuring system. For example, the term roughly can mean that the value is within 1 or more than 1 standard deviation, as shown by practice in this field. In cases where specific values are given in the application and the claims, then, unless otherwise indicated, the term approximately should be assumed, denoting being within the range of acceptable error for a particular value.

The terms individual, patient, or subject are used interchangeably. None of these terms requires or is limited to a situation characterized by supervision (eg, ongoing or occasional) by a healthcare professional (eg, physician, registered nurse, physician assistant, nurse, or hospice worker).

As used in the present invention, half-life is used in its usual sense as described in Goodman and Gillman's The Pharmaceutical Basis of Therapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and Alfred Gilman, eds., 6th ed. 1980 ). Briefly, this term is intended to cover a quantitative measure of the drug withdrawal period. The elimination of most drugs is exponential (i.e. follows first order kinetics) as drug concentrations usually do not reach those required to saturate the elimination process. The rate of an exponential process can be expressed by its rate constant, k, expressing a relative change per unit time, or by its half-life, t _1/2 , the time required for 50% completion of the process. The data units of the two constants are the time unit ^-1 and the time unit, respectively. The first order rate constant and the reaction half time are related simply (kxt _1/2 =0.693) and can be interchanged as appropriate. Because the first-order elimination kinetics dictate that a constant fraction of the drug is lost per unit time, the plot of the logarithm of drug concentration versus time is linear at any point in time after the initial phase of distribution (i.e., after drug absorption and distribution are completed ). The half time period for drug withdrawal can be accurately determined from such a schedule.

As used in the present invention, percent (%) amino acid sequence-to-sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular sequence, after aligning the sequences and inserting gaps, if necessary, to obtain the maximum percent sequence identity, and disregarding any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways known to those skilled in the art, for example using publicly available computer programs such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for determining the alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the compared sequences.

The term framework or FR residues (or regions) refers to those variable region residues that differ from CDRs or hypervariable region residues as defined herein. The human consensus framework region refers to the framework region that represents the most frequently occurring amino acid residue in the selection of VL or VH human immunoglobulin framework sequences.

As used in the present invention, the term variable region or variable domain

- 15 042731 refers to the fact that the sequences of certain parts of the variable domains differ significantly among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments, called complementarity determining regions (CDRs) or hypervariable regions, in the variable domains of both the light chain and the heavy chain. The more highly conserved portions of variable domains are called framework (FR). Each of the naturally occurring heavy and light chain variable domains contains four FR regions largely adopting a P-layer configuration connected by three CDRs that form loops connecting and in some cases part of the P-layer structure. The CDRs in each strand are co-located in close proximity to the FR regions and, together with the CDRs of the other strand, participate in the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but they have various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. Variable domain residue numbering as in Kabat or amino acid position numbering as in Kabat, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains for antibody data collection at Sequences of Proteins of Immunological Interest, 5th Ed . Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to a truncation or insertion in the FR or CDR of the variable domain.

For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 H2 and inserted residues (e.g., residues 82a, 82b and 82c, etc. according to Kabat) after residue 82 of the FR heavy chain. Kabat residue numbering can be determined for given antibodies by aligning regions of antibody sequence homology with a standard Kabat numbered sequence. It is not intended that the CDRs of the present invention necessarily follow the Kabat numbering system.

Used in the present invention, the term binding affinity refers to the affinity of the proteins described in the invention, in relation to their binding targets, and is expressed numerically using Kd values. If two or more proteins are indicated to have comparable binding affinities for their binding targets, then the Kd values for binding of the corresponding proteins to their binding targets are ±2-fold different from each other. If two or more proteins are indicated to have comparable binding affinities for a single binding target, then the Kd values for binding of the respective proteins to said single binding target are ±2-fold different from each other. If a protein is indicated to bind to two or more targets with comparable binding affinities, then the Kd values for said protein to bind to two or more targets are ±2-fold different from each other. In general, a higher Kd value corresponds to a weaker binding. In some embodiments, Kd is measured using a radiolabelled antigen (RIA) binding assay or surface plasmon resonance using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ). In some embodiments, the binding rate or association rate or connection rate or k _on and the dissociation rate or break rate or disconnection rate or kf are also determined by a surface plasmon resonance method using BIAcore™-2000 or a BIAcore™-3000 systems (BIAcore , Inc., Piscataway, NJ). In further embodiments, Kd, kon, and ko _ff are measured using Octet® Systems (Pall Life Sciences).

The present invention describes proteins containing a single chain variable fragment that binds to CD3, pharmaceutical compositions, as well as nucleic acids, recombinant expression vectors and host cells for obtaining such proteins containing a single chain variable fragment that binds to CD3. Also provided are methods of using the disclosed proteins comprising a CD3-binding single chain variable fragment to prevent and/or treat diseases, conditions and disorders. In some embodiments, proteins containing a CD3-binding single-chain variable fragment are capable of specifically binding to a CD3 domain as well as a target antigen and a half-life-extending domain, such as a human serum albumin-binding (HSA) single-domain antibody.

CD3 binding domain.

The specificity of the T cell response is mediated by antigen recognition (displayed in the context of the major histocompatibility complex, MHC) by the T cell receptor complex. As part of the com

The CD3 T-cell receptor plex is a protein complex that includes a CD3y chain (gamma), a CD3δ chain (delta), and two CD3ε chains (epsilon) that are present on the cell surface. CD3 associates with the α (alpha) and β (beta) chains of the T cell receptor (TCR) as well as the CD3 ζ (zeta) chain, which together form the T cell receptor complex. Clustering of CD3 on T cells, for example by immobilized anti-CD3 antibodies, results in T cell activation similar to but independent of T cell receptor specificity.

In one aspect, the CD3 binding single chain variable fragment proteins described herein comprise a domain that specifically binds to CD3. In one aspect, the CD3 binding single chain variable fragment proteins described herein comprise a domain that specifically binds to human CD3. In one aspect, the CD3 binding single chain variable fragment proteins described herein comprise a domain that specifically binds to cynomolgus monkey CD3. In one aspect, the CD3 binding single chain variable fragment proteins described herein comprise a domain that binds to human CD3 and cynomolgus CD3. In some embodiments, proteins described herein comprising a CD3-binding single-chain variable fragment contain a domain that specifically binds to CD3y. In some embodiments, proteins described herein comprising a CD3-binding single-chain variable fragment contain a domain that specifically binds to CD3δ. In some embodiments, proteins described herein comprising a CD3-binding single-chain variable fragment contain a domain that specifically binds to CD3ε.

In another aspect, a multispecific binding protein is provided, comprising a protein comprising a single chain variable fragment that binds to CD3, in accordance with the present invention. In some embodiments, a multispecific protein comprising a CD3-binding single chain variable fragment-containing protein of the present invention binds specifically to a T cell receptor (TCR). In some instances, a multispecific protein comprising a CD3-binding single-chain variable fragment-containing protein of the present invention binds to the α-chain of a TCR. In certain instances, a multispecific protein comprising a CD3-binding single-chain variable fragment-containing protein of the present invention binds to the β-chain of a TCR.

In some embodiments, the CD3 binding domain of the proteins described herein, comprising a CD3-binding single-chain variable fragment, exhibits not only strong CD3-binding affinity for human CD3, but also excellent cross-reactivity with the corresponding cynomolgus monkey CD3 proteins. In some instances, the CD3-binding domain of proteins containing a CD3-binding single-chain variable fragment is cross-reactive with cynomolgus monkey CD3. In certain cases, the Kd value for binding to human CD3 (hKd) is about the same as the Kd value for binding to cynomolgus monkey CD3 (cKd). In some cases, the ratio of hKd to cKd (hKd:Kd) ranges from about 20:1 to about 1:2.

In some embodiments, the CD3-binding domain of a protein containing a CD3-binding single-chain variable fragment can be any domain that binds to CD3, including, but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, humanized antibody. In some cases, it is advantageous that the CD3 binding domain be derived from the same species that will eventually use the CD3 binding single chain variable fragment containing protein. For example, for use in humans, it may be advantageous for the CD3 binding domain of the protein containing the CD3 binding single chain variable fragment to contain human or humanized residues from the antigen binding domain of an antibody or antibody fragment.

Thus, in one aspect, the antigen binding domain comprises a humanized or human antibody or antibody fragment, or a mouse antibody or mouse antibody fragment. In one embodiment, the humanized or human anti-CD3 binding domain comprises one or more (e.g., all three) of Light Chain Complementarity Determining Region 1 (LC CDR1), Light Chain Complementarity Determining Region 2 (LC CDR2), and Complementarity Determining Region 3 light chain (LC CDR3) of the humanized or human anti-CD3 binding domain described in the present invention, and/or one or more (e.g., all three) of the heavy chain complementarity determining region 1 (HC CDR1), complementarity determining region 2 of the heavy chain (HC CDR2) and complementarity determining region 3 of the heavy chain (HC CDR3) of the humanized or human anti-CD3-binding domain,

- 17 042731 described in the present invention, for example, a humanized or human anti-CD3 binding domain containing one or more, for example, all three LC CDRs and one or more, for example, all three HC CDRs.

In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human light chain variable region specific for CD3, wherein the CD3 specific light chain variable region contains human or non-human light chain CDR sequences in a human light chain framework region. In certain instances, the light chain framework is the λ (lambda) light chain framework. In other instances, the light chain framework is the k (kappa) light chain framework.

In some embodiments, the humanized or human anti-CD3 binding domain comprises a humanized or human heavy chain variable region specific for CD3, wherein the heavy chain variable region specific for CD3 comprises human or non-human heavy chain CDR sequences in a human heavy chain framework region.

In certain cases, the complementarity-determining regions of the heavy chain and/or light chain are derived from known anti-CD3 antibodies, such as e.g. SP34, TR-66 or X35-3, VIT3, WMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b , 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

In one embodiment, the anti-CD3 binding domain is a single chain variable fragment (scFv) containing a light chain and a heavy chain with the amino acid sequence provided in the present invention. As used herein, the term single chain variable fragment or scFv refers to an antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are adjacently linked via a short, flexible polypeptide linker. , and are capable of being expressed as a single polypeptide chain, while scFv retains the specificity of the intact antibody from which it is derived. In one embodiment, the anti-CD3 binding domain comprises: a light chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions) amino acid sequence of the variable region of the light chain presented in the present invention, or a sequence having 95-99% identity with the amino acid sequence presented in the present invention; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two, or three modifications (e.g., substitutions) but no more than 30, 20, or 10 modifications (e.g., substitutions) in the amino acid sequence of the heavy chain variable region presented in the present invention, or a sequence having 95-99% identity with the amino acid sequence presented in the present invention. In one embodiment, the humanized or human anti-CD3 binding domain is scFv and the light chain variable region containing the amino acid sequence described herein is fused to the heavy chain variable region containing the amino acid sequence described herein by scFv linker. The light chain variable region and the heavy chain variable region of the scFv may be located, for example, in any of the following directions: light chain variable region-scFv-linker-heavy chain variable region or heavy chain variable region-scFv-linker-light chain variable region.

In some cases, scFvs that bind to CD3 are generated according to known methods. For example, scFv molecules can be made by linking the VH and VL regions together using flexible polypeptide linkers. The scFv molecules contain an scFv linker (eg, a Ser-Gly linker) with an optimized length and/or amino acid composition. Accordingly, in some embodiments, the length of the scFv linker is such that the VH or VL domain can be linked intermolecularly to another variable domain to form a CD3 binding site. In certain embodiments, such scFv linkers are short, i. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Thus, in some instances, scFv linkers are composed of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the scFv linker is a peptide bond. In some embodiments, these scFv linkers consist of about 3-15, eg 8, 10, or 15 contiguous amino acid residues. With regard to the amino acid composition of the scFv linkers, peptides have been selected that provide flexibility, do not interfere with variable domains, and also allow for interchain folding to combine two variable domains to form a functional CD3 binding site. For example, scFvlinkers containing glycine and serine residues typically confer protease resistance. IN

- 18 042731 In some embodiments, linkers in scFv contain glycine and serine residues. The amino acid sequence of scFv linkers can be optimized, for example, using phage display techniques to improve CD3 binding and generate large amounts of scFv. Examples of peptidic scFv linkers suitable for linking a light chain variable domain and a heavy chain variable domain in scFv include, but are not limited to, (GS) _n (SEQ ID NO: 96), (GGS) _n (SEQ ID NO: 97 ), (GGGS)n(SEQ ID NO: 98), (GGSG)n(SEQ ID NO: 99), (GGSGG)n(SEQ ID NO: 100), (GGGGS)n(SEQ ID NO: 101), (GGGGG)n (SEQ ID NO: 102) or (GGG)n (SEQ ID NO: 103), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, implementation of the linker in scFv can be (GGGGS) ₄ (SEQ ID NO: 104) or (GGGGS) ₃ (SEQ ID NO: 1). Changing the length of the linker can maintain or increase activity, resulting in higher performance in activity assays.

In some embodiments, the CD3-binding domain of a protein containing a CD3-binding single-chain variable fragment has an affinity for CD3 on CD3-expressing cells with a Kd value of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less , 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In some embodiments, the CD3 binding domain of a protein containing a CD3-binding single-chain variable fragment has an affinity for CD3ε, γ, or δ with a K _d value of 1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less , 80 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, or 0.5 nM or less. In other embodiments, the CD3-binding domain of the protein containing the CD3-binding single-chain variable fragment has a low affinity for CD3, ie, about 100 nM or higher.

In certain embodiments, proteins described herein comprising a CD3-binding single chain variable fragment bind to human CD3 with human Kd (hKd) and cynomolgus monkey CD3 with cynomolgus monkey Kd (cKd). In some embodiments, the hKd and cKd values are in the range of about 1 nM to about 2 nM, about 3 nM to about 5 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nM to about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nM to about 160 nM , from about 170 nM to about 200 nM, from about 210 nM to about 250 nM, from about 260 nM to about 300 nM.

In some embodiments, the hKd and cKd values of proteins comprising a CD3-binding single-chain variable fragment are about the same as the Kd value of a CD3 binding protein having the sequence set forth in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of the proteins containing a single chain variable fragment that binds to CD3 is about 1.1-1.5 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of the proteins containing a single chain variable fragment that binds to CD3 is about 1.5-2 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins containing single chain variable fragment that binds to CD3 is about 2.5-3 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins containing a single chain variable the CD3 binding fragment is about 3-5 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins containing a single chain variable fragment that binds to CD3, about 6-15 times higher than the Kd of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins containing a CD3-binding single-chain variable fragment are about 15 -20 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins containing a CD3 binding single chain variable fragment are about 20-50 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins containing a CD3 binding single chain variable fragment are about 55-70 times higher than the Kd value A CD3 binding protein having the sequence set forth in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins comprising a CD3 binding single chain variable fragment are about 75-100 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22. In some embodiments, the hKd and cKd values of proteins containing a CD3 binding single chain variable fragment are about 120-200 times higher than the Kd value of a CD3 binding protein having the sequence shown in SEQ ID NO: 22.

In some embodiments, the ratio between hKd and cKd (hKd:Kd) ranges from about 20:1 to about 1:2. Affinity for binding to CD3 can be determined, for example, by

- 19 042731 the ability of the CD3-binding single-chain variable fragment-containing protein itself or its CD3-binding domain to bind to CD3 deposited on the assay plate; displayed on the microbial cell surface; in solution; etc. The binding activity of the CD3-binding single-chain variable fragment-containing protein itself or its CD3-binding domain according to the present invention to CD3 can be analyzed by immobilizing the ligand (e.g., CD3) or the CD3-binding single-chain variable fragment-containing protein itself) , or its CD3-binding domain on a bead, scaffold, cage, etc. Agents in an appropriate buffer may be added and binding partners may be incubated for a specified period of time at a given temperature. After washes to remove unbound material, bound protein can be released using, for example, SDS, high pH buffers, and the like, and analyzed, for example, using Surface Plasmon Resonance (SPR).

In some embodiments, the CD3-binding single-chain variable fragment-containing protein has an amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 94) and SEQ ID NO: 95.

In some embodiments, the CD3-binding single chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 8, wherein the hKd is about 3.8 nM and the cKd is about 3.5 nM. In some embodiments, the CD3-binding single chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 9, with an hKd of about 4.1 nM and with a cKd of about 3.4 nM. In some embodiments, the CD3-binding single chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 10, wherein hKd is about 4.3 nM and cKd is about 4.2 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 11, wherein the hKd is about 4.7 nM and the cKd is about 4.9 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 12, with an hKd of about 6.4 nM and with a cKd of about 6.6 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 13, wherein the hKd is about 8 nM and the cKd is about 6.6 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 14, wherein the hKd is about 20 nM and the cKd is about 17 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 15, with an hKd of about 37 nM and with a cKd of about 30 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 16, wherein the hKd is about 14 nM and the cKd is about 13 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 17, wherein the hKd is about 50 nM and the cKd is about 47 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 18, wherein the hKd is about 16 nM and the cKd is about 16 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 19, wherein the hKd is about 46 nM and the cKd is about 43 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 20, wherein the hKd is about 18 nM and the cKd is about 17 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 21, wherein the hKd is about 133 nM and the cKd is about 134 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 94, wherein the hKd is about 117 nM and the cKd is about 115 nM. In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 95, with an hKd of about 109 pM and with a cKd of about 103 nM.

- 20 042731

In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 8, wherein the hKd and cKd values are approximately the same as the Kd value relative to CD3 of the protein that has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 9, wherein the hKd and cKd values are approximately the same as the Kd value relative to CD3 of the protein that has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 10, wherein the hKd and cKd values are about the same as the Kd value relative to CD3 of the protein that has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 11, wherein the hKd and cKd values are about the same as the Kd value relative to CD3 of the protein that has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 12, wherein the hKd and cKd values are about the same as the Kd value relative to CD3 of the protein that has the sequence presented in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 13, wherein the hKd and cKd values are about the same as the Kd value relative to CD3 of the protein that has the sequence presented in wt antiCD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 14, with hKd and cKd values about 3-5 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 15, with hKd and cKd values about 3-5 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 16, with hKd and cKd values about 3-5 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt nachi-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 17, wherein the hKd and cKd values are about 6-15 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 18, with hKd and cKd values about 3-5 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt antiCD3 (SEQ ID NO: 22). In some embodiments, the CD3-binding single-chain variable fragment-containing protein has the amino acid sequence shown in SEQ ID NO: 19 (2A4), with hKd and cKd values about 6-15 times higher than the Kd value relative to CD3 protein which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 20, with hKd and cKd values about 3-5 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 21, with hKd and cKd values about 20-50 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a single chain variable fragment that binds to CD3 has the amino acid sequence shown in SEQ ID NO: 94, with hKd and cKd values about 20-50 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22). In some embodiments, a protein containing a CD3-binding single-chain variable fragment has the amino acid sequence shown in SEQ ID NO: 95, wherein the hKd and cKd values are about 20-50 times higher than the Kd value relative to the CD3 protein, which has the sequence shown in wt anti-CD3 (SEQ ID NO: 22).

- 21 042731

A domain that prolongs the half-life.

Human serum albumin (HSA) (molecular weight ~67 kDa) is the most abundant protein in plasma, present at about 50 mg/ml (600 µM) and has a half-life of about 20 days in the body person. HSA serves to maintain blood plasma pH, contributes to the maintenance of colloidal arterial pressure, functions as a carrier for many metabolites and fatty acids, and serves as a major protein for plasma drug transport.

Non-covalent association with albumin lengthens the half-life of short-lived proteins. For example, recombinant fusion of an albumin-binding domain to a Fab fragment resulted in 25-fold and 58-fold in vivo clearance and a 26-fold and 37-fold prolongation of the half-life when intravenously administered to mice and rabbits, respectively, compared with administration of the Fab fragment alone. In another example, when insulin is acylated with fatty acids to improve binding to albumin, a long-lasting effect was observed when administered subcutaneously in rabbits or pigs. Taken together, the data from these studies demonstrate an association between albumin binding and sustained release.

In one aspect, a multispecific binding protein is provided, comprising a protein comprising a CD3-binding single-chain variable fragment according to the present invention, and further comprising a half-life prolonging domain, e.g., a domain that specifically binds to serum albumin. In some embodiments, the serum albumin-binding domain of a protein containing a CD3-binding single-chain variable fragment can be any domain that binds to serum albumin, including, but not limited to, domains from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, humanized antibody. In some embodiments, the serum albumin binding domain is single chain variable fragments (scFv), a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) from a camel-derived single domain antibody, or antigen-binding fragments of HSA-binding antibodies, such as Fab, Fab', F(ab)2 and Fv fragments, fragments consisting of one or more CDRs, single-chain antibodies (for example, single-chain Fv fragments (scFv)), disulfide stabilized ( dsFv) Fv fragments, heteroconjugate antibodies (e.g. bispecific antibodies), pFv fragments, heavy chain monomers or dimers, light chain monomers or dimers, and dimers consisting of one heavy chain and one light chain, peptide, ligand or small molecular weight group, specific to serum albumin. In certain embodiments, the HSA binding domain is a single domain antibody. In other embodiments, the serum albumin binding domain is a peptide. In other embodiments, the serum albumin binding domain is a small molecular weight group. It is assumed that the serum albumin-binding domain of the multispecific binding protein, which is a protein containing a single-chain variable fragment that binds to CD3, has a sufficiently small size and is not more than 25 kDa, not more than 20 kDa, not more than 15 kDa, or not more than 10 kDa, in some embodiments. In some cases, the serum albumin binding domain is 5 kDa or less when it is a peptide or a small molecular weight group.

The half-life extension domain of the CD3-binding single-chain variable fragment-containing protein provides a change in the pharmacodynamics and pharmacokinetics of the CD3-binding single-chain variable fragment-containing protein itself. As mentioned above, a domain that provides an extension of the half-life, prolongs the half-life. The half-life-prolonging domain also alters pharmacodynamic properties, including altering the distribution, penetration, and diffusion of the CD3-binding single-chain variable fragment-containing protein in tissue. In some embodiments, the half-life extension domain provides improved tissue targeting (including tumor), tissue distribution, tissue penetration, intra-tissue diffusion, and increased efficiency compared to a protein without a half-life extension domain. In one embodiment, the therapeutic methods effectively and efficiently use a reduced amount of a multispecific binding protein, which is a protein containing a single chain variable fragment that binds to CD3, resulting in a reduction in side effects, such as a decrease in non-tumor cytotoxicity.

In addition, the binding affinity of the half-life-prolonging domain can be chosen to correspond to a certain half-life in a particular multispecific binding protein, which is a protein containing a single-chain variable fragment that binds to CD3. Thus, in some embodiments, the half-life extension domain has a high binding affinity. In other embodiments, the implementation of the domain that provides an extension of the half-life

- 22 042731 denia, has an average binding affinity. In other embodiments, the half-life extension domain has low or marginal binding affinity. Examples of binding affinity include Kd concentrations of 10 nM or less (high), ranging from 10 nM to 100 nM (medium), and greater than 100 nM (low). As mentioned above, binding affinities for serum albumin are determined using known methods such as surface plasmon resonance (SPR).

The binding domain of the target antigen.

In addition to the CD3 described and the half-life prolonging domain, the multispecific binding proteins described herein in certain embodiments also contain a domain that binds to a target antigen. The target antigen is involved in a disease, disorder, or condition and/or associated with the disease, disorder, or condition. In particular, the target antigen is associated with a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease, or host-versus-graft disease. In some embodiments, the target antigen is a tumor antigen expressed on a tumor cell. Alternatively, in some embodiments, the target antigen binds to a pathogen, such as a virus or bacterium.

In some embodiments, the target antigen is a cell surface molecule such as a protein, lipid, or polysaccharide. In some embodiments, the target antigen is on a tumor cell, a virus-infected cell, a bacterially infected cell, an injured red blood cell, an arterial plaque cell, or a fibrous tissue cell.

The design of the multispecific binding protein, which is a protein described in the present invention, containing a single-chain variable fragment that binds to CD3, allows the target antigen binding domain to be universal in the sense that the target antigen binding domain can be any type of binding domain including, but not limited to, monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody domains. In some embodiments, the target antigen binding domain is single chain variable fragments (scFv), a single domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) from a camel-derived single domain antibody . In other embodiments, the target antigen binding domain is a non-Ig binding domain, i.e., an antibody mimetic such as anticalins, affilins, affitel molecules, affimers, affitins, alphabodies, avimers, DARPin ankyrin repeat proteins, finomers, peptides with the Kunitz domain and the monobody. In other embodiments, the target antigen binding domain is a ligand or peptide that binds or associates with the target antigen. In other embodiments, the target antigen binding domain is nottin. In other embodiments, the target antigen binding domain is a small molecular weight group.

Modifications of proteins containing a single-chain variable fragment that binds to CD3.

The proteins described herein comprising a CD3-binding single-chain variable fragment include derivatives or analogs in which (i) an amino acid is replaced by an amino acid residue that is not the amino acid residue encoded by the genetic code, (ii) the mature polypeptide is fused to another compound, such as polyethylene glycol, or (iii) additional amino acids fused to the protein, such as a leader sequence or a secretory sequence, or a sequence to block the immunogenic domain and/or to purify the protein.

Representative modifications include, but are not limited to, acetylation, acylation, ADPribosylation, amidation, flavin covalent attachment, heme covalent attachment, nucleotide or nucleotide derivative covalent attachment, lipid or lipid derivative covalent attachment, phosphatidylinositol covalent attachment, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI-anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transport mediated sulfation RNA is the addition of amino acids to proteins, such as arginylation and ubiquitination.

Modifications are made anywhere in the proteins described herein containing a CD3-binding single chain variable fragment, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Some common peptide modifications that are useful for modifying proteins containing a CD3-binding single-chain variable fragment include glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, blocking of an amino or carboxyl group, or both. in the polypeptide, through covalent modification, and ADP-ribosylation.

Polynucleotides encoding proteins containing a single-chain variable fragment that binds to CD3.

The present invention also relates, in some embodiments, to polynucleotide molecules encoding a CD3-binding single-chain variable fragment-containing protein or a multispecific binding protein comprising a CD3-binding single-chain variable fragment-containing protein according to the present invention. In some embodiments, the polynucleotide molecules are provided as a DNA construct. In other embodiments, the polynucleotide molecules are provided as a messenger RNA transcript.

Polynucleotide molecules are constructed by known methods, for example, by combining genes encoding three binding domains, either separated by peptide linkers or, in other embodiments, directly linked by a peptide bond, into a single genetic construct, operably linked to a suitable promoter and optionally a suitable transcription terminator, and their expression in bacteria or other suitable expression systems, for example in CHO cells. In embodiments in which the binding domain of the target antigen is a small molecular weight group, the polynucleotides contain genes encoding a CD3 binding domain and a half-life prolonging domain. In embodiments where the half-life prolonging domain is a small molecular weight group, the polynucleotides contain genes encoding domains that bind to CD3 and the target antigen. Depending on the vector system and host used, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. The promoter is chosen such that it drives the expression of the polynucleotide in the appropriate host cell.

In some embodiments, the polynucleotide is inserted into a vector, preferably an expression vector, which is a further embodiment. This recombinant vector can be constructed in accordance with known methods. Vectors of particular interest include plasmids, phagemids, phage derivatives, viruses (eg, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, and the like), and cosmids.

Various vector/host expression systems can be used to serve as a carrier and be used to express a polynucleotide encoding a polypeptide of the described protein containing a single chain variable fragment that binds to CD3. Examples of expression vectors for expression in E. coli are pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27) or pcDNA5 (Invitrogen) for expression in mammalian cells, PICHIAPINK™ Yeast Expression Systems (Invitrogen), BACUVANCE™ Baculovirus Expression System (GenScript).

Thus, proteins containing a CD3-binding single-chain variable fragment as described herein are, in some embodiments, produced by introducing a vector encoding a protein as described above into a host cell and culturing said host cell under conditions at whose protein domains are expressed can be isolated and, if necessary, further purified.

Obtaining proteins containing a single-chain variable fragment that binds to CD3.

In some embodiments, the present invention discloses a method for producing a protein containing a single chain variable fragment that binds to CD3. In some embodiments, the method includes culturing a host transformed or transfected with a vector containing a nucleic acid sequence encoding a CD3-binding single-chain variable fragment-containing protein under conditions that allow expression of the CD3-binding single-chain variable fragment-containing protein, and isolating and purification of the obtained protein from the culture.

In a further embodiment, a method is provided to improve one or more properties, such as affinity, stability, high temperature stability, cross-reactivity, and so on. a CD3-binding single-chain variable fragment-containing protein; and/or multispecific binding proteins comprising a CD3-binding single-chain variable fragment-containing protein as described herein compared to a reference binding compound. In some embodiments, a plurality of single substitution libraries are provided, each corresponding to a different domain or amino acid segment of the CD3-binding single chain variable fragment-containing protein or reference binding compound, such that each member of the single substitution library encodes only one amino acid substitution in its corresponding domain or amino acid segment. (This allows all potential substitutions in a large protein or protein-binding site to be explored with several small libraries.) binding connection. The nucleotide sequences of the various single substitution libraries overlap with the nucleotide sequences of at least one other single substitution library. In some embodiments, a plurality of single substitution libraries are designed such that each member overlaps with each member of each single substitution library encoding an adjacent domain.

Binding compounds expressed from such single substitution libraries are selected separately to obtain a subset of variants in each library that have at least the same properties as the reference binding compound, and the resulting library is reduced in size. (That is, the number of nucleic acids encoding the selected set of binding compounds is less than the number of nucleic acids encoding members of the original library with single substitutions). Such properties include, but are not limited to, affinity for the target compound, stability to various conditions such as heat, high or low pH, enzymatic degradation, cross-reactivity with other proteins, and the like. Selected compounds from each library with single substitutions are referred to herein interchangeably as candidate pre-compounds or pre-candidate proteins. Nucleic acid sequences encoding candidate pre-compounds from individual libraries with single substitutions are then shuffled in PCR to create a shuffled library using PCR-based gene shuffling techniques.

The present invention describes an exemplary procedure for conducting a screening process. Candidate pre-libraries were generated from single substitution libraries and selected for binding to the target protein(s), after which the pre-candidate libraries were shuffled to produce a library of nucleic acids encoding candidate compounds, which in turn were cloned into the appropriate expression a vector such as a phagemid expression system. Candidate phage-expressing compounds were then subjected to one or more rounds of selection to improve desired properties such as binding affinity for the target molecule. Target molecules can be adsorbed or otherwise attached to the surface of a well or other reaction container, or target molecules can be derivatized using a binding moiety, such as biotin, which, after incubation with candidate binding compounds, can be captured by a complementary moiety, such as streptavidin associated with particles, such as magnetic particles, for washing. In exemplary selection modes, candidate binding compounds are subjected to a lengthy washing step, thus only candidate compounds with very low rates of dissociation from the target molecule are selected. Exemplary wash times for such embodiments are at least 8 hours; or, in other embodiments, at least 24 hours; or, in other embodiments, at least 48 hours; or, in other embodiments, at least 72 hours. Selected clones after selection were amplified and subjected to an additional round of selection or analyzed, for example, by sequencing and by performing comparative measurements of binding affinity, for example, using ELISA, binding by surface plasmon resonance, biolayer interferometry (e.g. Octet system, ForteBio, Menlo Park, CA) or the like.

In some embodiments, the process is performed to identify one or more CD3-binding single-chain variable fragment-containing proteins and/or a multispecific binding protein comprising a CD3-binding single-chain variable fragment-containing protein with improved thermal stability, improved cross-reactivity in against a selected set of binding targets compared to a reference CD3 binding protein, such as the protein having the amino acid sequence of SEQ ID NO: 22. Libraries with single substitutions were generated by changing codons in the VH and VL regions of the reference CD3 binding protein, including codons in framework regions and in CDRs; in another embodiment, the codon-changed locations contain the heavy and light chain CDRs of the reference CD3 binding protein, or a subset of such CDRs, eg, CDR1 only, CDR2 only, CDR3 only, or pairs thereof. In another embodiment, localizations where codons have been changed occur only in the framework regions. In some embodiments, the library contains only one codon change from the reference CD3 binding protein, only in the framework regions of both VH and VL, numbered in the range 10 to 250. and light chains of the reference CD3 binding protein, or a subset of such CD3s. In another embodiment, the number of locations where the codons of the VH and VL coding regions have been changed is in the range of 10 to 250, eg up to 100 locations are in the framework region. After obtaining a library with single substitutions, as described above, the following steps were performed: (a) expression separately of each

- 25,042,731 single substitution members of each library as a preliminary candidate protein; (b) selecting members of each library with single substitutions that encode tentative candidate proteins that bind to a binding partner other than the original binding target (eg, the desired cross-reactive target(s)); (c) shuffling the members of the selected libraries into PCR to obtain a combinatorial shuffled library; (d) expression of members of the shuffled library as candidate CD3-binding proteins; and (e) selecting members of the shuffled library one or more times for candidate CD3 binding proteins that bind to the original binding partner, and (f) further selecting candidate proteins to bind to the desired cross-reactive target(s), thereby providing a nucleic acid-encoded CD3 binding protein with increased cross-reactivity for one or more substances to the reference CD3 binding protein without loss of affinity for the parent ligand. In additional embodiments, the method can be carried out to produce a protein containing a CD3-binding single-chain variable fragment with reduced reactivity with the selected cross-reactive substance(s) or compound(s) or epitope(s) by replacing the step ( f) next step: depleting the candidate binding compounds one or more times from a subset of candidate protein containing a CD3-binding single-chain variable fragment that binds to the unwanted cross-reacting compound.

pharmaceutical compositions.

In addition, in some embodiments, pharmaceutical compositions are provided comprising a CD3-binding single-chain variable fragment-containing protein described herein, a vector containing a polynucleotide encoding a CD3-binding single-chain variable fragment-containing protein polypeptide, or a host cell, transformed with this vector, and at least one pharmaceutically acceptable carrier. The term pharmaceutically acceptable carrier includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, various types of wetting agents, sterile solutions, and the like. Such carriers may be prepared by conventional means and may be administered to the subject at a suitable dose. Preferably the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifiers and dispersants. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents.

In some embodiments of pharmaceutical compositions, a CD3-binding single-chain variable fragment-comprising protein described herein is encapsulated in nanoparticles. In some embodiments, the nanoparticles are fullerenes, liquid crystals, liposomes, quantum dots, superparamagnetic nanoparticles, dendrimers, or nanorods. In other embodiments of the pharmaceutical compositions, a CD3-binding single-chain variable fragment-containing protein is attached to liposomes. In some cases, the protein containing the CD3 single chain variable fragment is conjugated to the surface of the liposomes. In some cases, the protein containing the CD3 single chain variable fragment is encapsulated in a liposome envelope. In some cases, the liposome is a cationic liposome.

Described in the present invention, proteins containing a single-chain variable fragment that binds to CD3, are considered for use as a drug. The introduction is carried out in various ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, local or intradermal administration. In some embodiments, the route of administration depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. Dosing for any patient depends on many factors, including patient size, body surface area, age, sex, particular compound to be administered, time and route of administration, type of therapy, general health, and other drugs administered concomitantly. An effective dose refers to amounts of the active ingredient that are sufficient to affect the course and severity of the disease, resulting in a reduction or remission of such pathology and can be determined using known methods.

Methods of treatment.

Also in some embodiments, the present invention provides methods and uses for stimulating the immune system of an individual in need thereof, comprising administering a CD3-binding single chain variable fragment-containing protein described herein. In some cases, the introduction of the protein described in the present invention,

- 26 042731 containing a single-chain variable fragment that binds to CD3, induces and/or maintains cytotoxicity against a cell expressing the target antigen. In some instances, the cell expressing the target antigen is a cancer or tumor cell, a virus-infected cell, a bacterially infected cell, an autoreactive T or B cell, damaged red blood cells, arterial plaque, or fibrous tissue.

The present invention also provides methods and uses for the treatment of a disease, disorder or condition associated with a target antigen, comprising administering to an individual in need thereof a CD3-binding single chain variable fragment-containing protein described herein. Diseases, disorders, or conditions associated with the target antigen include, but are not limited to, viral infection, bacterial infection, autoimmune diseases, transplant rejection, atherosclerosis, or fibrosis. In other embodiments, the disease, disorder, or condition associated with the target antigen is a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, a graft-versus-host disease, or host-versus-graft disease. In one embodiment, the disease, disorder, or condition associated with the target antigen is cancer. In one case, the cancer is a hematological cancer. In another case, the cancer is a cancer with a solid tumor.

As used in some embodiments in the present invention, the term treated or treated or treated refers to therapeutic treatment, the purpose of which is to slow down (reduce) an undesirable physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes described in the present invention, beneficial or desirable clinical results include, but are not limited to, symptomatic relief; reducing the degree of the condition, disorder or disease; stabilization (ie, no deterioration) of the condition, disorder, or disease; delaying the onset or slowing the progression of the condition, disorder or disease; radical improvement of the condition, disorder or disease state; and remission (partial or complete), whether detectable or undetectable, or alleviation or amelioration of a condition, disorder or disease. Treatment includes identifying a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to expected survival if no treatment was received. In other embodiments, treating or treating or being treated refers to preventive measures the purpose of which is to delay the onset or reduce the severity of an undesired physiological condition, disorder, or disease, for example, in a person who is predisposed to the disease (for example, a person who carries a genetic marker for a disease such as breast cancer).

In some embodiments of the methods described herein, CD3 binding single chain variable fragment containing proteins are administered in combination with an agent for the treatment of a particular disease, disorder or condition. Means include, but are not limited to, therapies including antibodies, small molecules (e.g., chemotherapy drugs), hormones (steroid, peptide, etc.), radiotherapy (gamma rays, x-rays and/or targeted delivery of radioisotopes, microwave ovens , UV radiation, etc.), gene therapy (eg, antisense drugs, antiretroviral therapy, etc.), and other immunotherapies. In some embodiments, proteins containing a CD3-binding single-chain variable fragment are administered in combination with antidiarrheals, antiemetics, analgesics, opioids, and/or non-steroidal anti-inflammatory drugs. In some embodiments, proteins containing a CD3-binding single-chain variable fragment are administered before, during, or after surgery.

Examples

Example 1: Identification of anti-CD3 scFv variants with different affinities for human CD3ε.

Characterization of the original anti-CD3ε phage.

The original anti-CD3ε showed good binding to biotin-CD3ε and low binding to biotin HSA (Fig. 1).

Anti-CDSε scFv phage libraries.

A single substitution library was provided for the heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3 domains. The amino acid residues that vary within each domain are illustrated in the shaded area in FIG. 2. The residues were changed one by one by NNN mutagenesis.

Selection of clones and determination of binding affinity.

Libraries with single substitutions were bound to biotinylated hu-CD3ε, washed, eluted and counted. Biotinylated cynoCD3 (cynomolgus macaque) was used as

- 27 042731 targets for the selection of the first round and washed for 4 hours after combinatorial binding of phages from two independent libraries (~2x selection). Biotinylated huCD3 (human) was used as a second round selection target and washed for 3 hours after both libraries were bound (<2x selection). Inserts introduced by PCR from the second round of selection were subcloned into the pcDNA3.4 His6 expression vector (His6 disclosed as SEQ ID NO: 105). 180 clones were selected and the DNA was purified, sequenced and transfected into Expi293. A panel of sixteen clones with an affinity range for human CD3ε was selected to more accurately determine K _d (FIG. 3).

Example 2: cytotoxicity assay.

A bispecific antibody directed against CD20 and CD3 containing the anti-CD3 scFv variant identified in Example 1 was evaluated in vitro for its mediation of T-cell dependent cytotoxicity to CD20+ target cells.

Fluorescently labeled CD20+ REC-1 cells (mantle cell lymphoma line, ATCC CRL3004) were incubated with isolated random donor PBMCs or CB15 T cells (standardized T cell line) as effector cells in the presence of a CD20-CD3 bispecific antibody containing a variant of the anti- CD3 scFv identified in Example 1. After incubation for 4 hours at 37° C. in a humidified incubator, the release of fluorescent dye from the target cells into the supernatant was determined with a spectrofluorimeter. Target cells incubated without the D20CD3 bispecific antibody containing the anti-CD3 scFv variant identified in Example 1 and target cells completely lysed with the addition of saponin at the end of incubation served as negative and positive controls, respectively.

Based on the measured remaining living target cells, the percentage of specific cell lysis was calculated according to the following formula:

[1-(number of live targets( _sample )/number of live targets( _{spontaneously} ))]x100%

Sigmoid dose-response curves and EC ₅₀ values were calculated by non-linear regression/fitting a 4-parameter logistic model using GraphPad software. The lysis values obtained for a given variant concentration were used to calculate sigmoidal dose-response curves by 4-parameter logistic model fit analysis using Prism software.

Example 3: Thermal stability of anti-CD3 scFv variants with different affinities for human CD3ε.

The hydrophobic exposure temperature (T _h ) of a protein corresponds to the inflection point derivative of the peak fluorescence of the dye and is known to correlate with the melting temperature (T _m ), which is an indication of protein stability. The aim of this study was to evaluate T _h for several anti-human CD3ε scFv variants.

Getting protein.

The anti-human CD3ε scFv binding domain sequences were cloned into the pcDNA3.4 vector (Invitrogen), preceded by a leader sequence and followed by a 6x Histidine tag sequence (SEQ ID NO: 105). Expi293F cells (Life Technologies A14527) were maintained in suspension in optimal growth flasks (Thomson) at 0.2 to 8 x 1E6 cells/ml in Expi 293 growth medium. Purified plasmid DNA was transfected into Expi293F cells according to the protocols of the Expi293 Expression System. Kit (Life Technologies, A14635) and kept for 4 to 6 days after transfection. The conditioned media were partially purified by affinity and demineralization chromatography. Anti-human CD3ε scFv proteins were concentrated in Amicon Ultra centrifugal filtration devices (EMD Millipore), loaded onto Superdex 200 (GE Healthcare) size exclusion chromatography medium, and eluted in neutral buffer containing vehicles. Collected fractions and final purity were assessed by SDS-PAGE and analytical SEC (size exclusion chromatography). The absorbance of the purified protein solutions was determined at 280 nm using SpectraMax M2 (Molecular Devices) and UV transparent 96-well plates (Corning 3635) and their concentrations were calculated from the molar extinction coefficients.

Differential scanning fluorimetry.

Purified anti-human CD3ε scFv proteins were diluted to concentrations ranging from 0.2 to 0.25 mg/ml along with 5x SYPRO orange dye (Life Technologies S6651) at a final concentration of 0.15% DMSO in neutral buffer containing excipients, in optical microplates with MicroAmp EnduraPlate adhesive film (Applied Biosystems 4483485 and 4311971). A plate containing diluted protein and dye mixtures was loaded onto a Fast real-time PCR instrument (Applied Biosytems) and subjected to a multi-stage temperature gradient from 25°C to 95°C. The temperature gradient consisted of holding for two minutes at each degree Celsius at an excitation wavelength of 500 nm, and the emission was recorded using a ROX filter. In FIG. 4 T _h in degrees Celsius is shown for several purified protein variants containing anti-human CD3ε scFv.

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SEO ID NO: Description AA Sequence 1 Linker GGGGSGGGGSGGGGS 2 NA CDR1 with options provisions GX1X2X3NX4YX5X6N 3 NA CDR2 with options provisions RIR S X ₇ Xs N X ₉ Y Hyu T Xts Y X12 DX _i3 V K 4 NA CDR3 with options provisions Η X14 NF X15 X16 s X171 SYWAX _i8 5 LC CDR1 with options positions Hu X20 X21X22 G X ₂ s V X ₂ 4 X25 G X ₂ 6 Y P N 6 LC CDR2 with options positions GX27 X ₂₈ X29 Xzo Xz1 R 7 LC CDR3 with provisions

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options Xzg LWY Xzz Ν Χ ₃₄ W X35 8 Ahth-CD3, clone 2B2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGL EWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKT EDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGG SGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAE YYCTLWYSNRWVFGGGTKLTVL 9 Ahth-CD3, clone 9F2 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNWVRQAPGKG LEWVARIRSKYNKYATYY AD S VKDRFTISRDD SKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYDNRWVFGGGTKLTVL 10 Ahth-CD3, clone 5A2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKG LEWVARIRSKYNNYATYY AD S VKDRFTISRDD SKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSHISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGYVTSGNYPNWVQ QKPGQAPRGLIGGTSFLAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWIFGGGTKLTVL eleven Ahth-CD3, clone 6A2 EVQLVESGGGLVQPGGSLKLSCAASGFMFNKYAMNWVRQAPGKG LEWVARIRSKSNNY ATYY AD S VKDRFTISRDD SKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSYISYWATWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQ QKPGQAPRGLIGGTKLLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNSWVFGGGTKLTVL 12 Ahth-CD3, 2D2 clone EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYKDSVKDRFTISRDDSKNTAYLQMNNLKT EDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGG SGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVVSGNYPNWVQ QKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWVFGGGTKLTVL 13 Ahth-CD3, clone 3F2 EVQLVESGGGLVQPGGSLKLSCAASGFTYNKYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADEVKDRFTISRDDSKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSKGAVTSGNYPNWV QQKPGQAPRGLIGGTKELAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCTLWYSNRWVFGGGTKLTVL 14 Ahth-CD3, clone 1A2 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNWVRQAPGKG LEWVARIRSKYNNYETYYADSVKDRFTISRDDSKNTAYLQMNNLK TEDTAVYYCVRHTNFGNSYISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQ

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QKPGQAPRGLIGGTYFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL 15 Ahth-CD3, clone 1C2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADAVKDRFTISRDDSKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSQISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTDGNYPNWV QQKPGQAPRGLIGGIKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL 16 Ahth-CD3, clone 2E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAVNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKT EDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGG SGGGGSQTVVTQEPSLTVSPGGTVTLTCGESTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKILAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWVFGGGTKLTVL 17 Ahth-CD3, clone 10E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYPMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKN EDTAVYYCVRHGNFNNSYISYWAYWGQGTLVTVSSGGGGSGGGG SGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTKGNYPNWVQ QKPGQAPRGLIGGTKMLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCALWYSNRWVFGGGTKLTVL 18 Ahth-CD3, clone 2H2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADEVKDRFTISRDDSKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVVSGNYPNWV QQKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL 19 Ahth-CD3, clone 2A4 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNWVRQAPGKG LEWVARIRSKYNNYATYY AD S VKDRFTISRDD SKNTAYLQMNNLK TEDTAVYYCVRHGNFGDSYISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTHGNYPNWV QQKPGQAPRGLIGGTKVLAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYSNRWVFGGGTKLTVL 20 Ahth-CD3, clone 10B2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNWVRQAPGKG LEWVARIRSGYNNYATYY AD S VKDRFTISRDD SKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSYTGAVTSGNYPNWV QQKPGQAPRGLIGGTKFNAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYANRWVFGGGTKLTVL 21 Ahth-CD3, clone 1G4 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNWVRQAPGKG LEWVARIRSKYNNYETYYADSVKDRFTISRDDSKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSLISYWAYWGQGTLVTVSSGGGGSGGG

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GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSSGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFGAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL 22 wt anti-pops EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKG LEWVARIRSKYNNYATYY AD S VKDRFTISRDD SKNTAYLQMNNLK TEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGG GSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL 23 wt anti-POPs NA CDR1 GFTFNKYAMN 24 wt anti-POPs HCCDR2 RIRSKYNNYATYYADSVK 25 wt anti-POPs NA CDR3 HGNFGNSYISYWAY 26 wt anti-POPs LC CDR1 GSSTGAVTSGNYPN 27 wt anti-POPs LC CDR2 GTKFLAP 28 wt anti-POPs LC CDR3 VLWYSNRWV 29 HC CDR1 option 1 GNTFNKYAMN thirty NS CDR1 option 2 GFEFNKYAMN 31 HC CDR1 option 3 GFMFNKYAMN 32 HC CDR1 option 4 GFTYNKYAMN 33 NS CDR1 option 5 GFTFNNYAMN 34 NS CDR1 option 6 GFTFNGYAMN 35 NS CDR1 option 7 GFTFNTYAMN 36 NS CDR1 option 8 GFTFNEYAMN 37 NS CDR1 option 9 GFTFNKYPMN 38 NS CDR1 option 10 GFTFNKYAVN 39 NS CDR1 option 11 GFTFNKYAIN 40 NS CDR1 option 12 GFTFNKYALN 41 NS CDR2 option 1 RIRSGYNNYATYYADSVK 42 NS CDR2 option 2 RIRSKSNNYATYYADSVK 43 NS CDR2 option 3 RIRSKYNKYATYYADSVK 44 NS CDR2 variant 4 RIRSKYNNYETYYADSVK 45 NS CDR2 variant 5 RIRSKYNNYATEYADSVK 46 NS CDR2 option 6 RIRSKYNNYATYYKDSVK 47 NS CDR2 variant 7 RIRSKYNNYATYYADEVK 48 NS CDR2 option 8 RIRSKYNNYATYYADAVK 49 NS CDR2 variant 9 RIRSKYNNYATYYADQVK 50 NS CDR2 variant 10 RIRSKYNNYATYYADDVK 51 NS CDR3 option 1 HANFGNSYISYWAY 52 NS CDR3 option 2 HTNFGNSYISYWAY 53 NS CDR3 option 3 HGNFNNSYISYWAY 54 NS CDR3 option 4 HGNFGD SYISYWAY

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55 NS CDR3 option 5 HGNFGNSHISYWAY 56 NS CDR3 option 6 HGNFGNSPISYWAY 57 NS CDR3 option 7 HGNFGNSQISYWAY 58 NS CDR3 option 8 HGNFGNSLISYWAY 59 NS CDR3 option 9 HGNFGNSGISYWAY 60 NS CDR3 option 10 HGNFGNSYISYWAT 61 LC CDR1 option 1 ASSTGAVTSGNYPN 62 LC CDR1 option 2 GESTGAVTSGNYPN 63 LC CDR1 option 3 GSYTGAVTSGNYPN 64 LC CDR1 option 4 GSSFGAVTSGNYPN 65 LC CDR1 option 5 GSSKGAVTSGNYPN 66 LC CDR1 option 6 GSSSGAVTSGNYPN 67 LC CDR1 option 7 GSSTGYVTSGNYPN 68 LC CDR1 option 8 GSTGAVVSGNYPN 69 LC CDR1 option 9 GSSTGAVTDGNYPN 70 LC CDR1 option 10 GSSTGAVTKGNYPN 71 LC CDR1 option 11 GSSTGAVTHGNYPN 72 LC CDR1 option 12 GSSTGAVTVGNYPN 73 LC CDR1 option 13 GSSTGAVTSGYYPN 74 LC CDR2 option 1 GIKFLAP 75 LC CDR2 option 2 GTEFLAP 76 LC CDR2 option 3 GTYFLAP 77 LC CDR2 option 4 GTSFLAP 78 LC CDR2 option 5 GTNFLAP 79 LC CDR2 option 6 GTKLLAP 80 LC CDR2 option 7 GTKELAP 81 LC CDR2 option 8 GTKILAP 82 LC CDR2 option 9 GTKMLAP 83 LC CDR2 option 10 GTKVLAP 84 LC CDR2 option 11 GTKFNAP 85 LC CDR2 option 12 GTKFGAP 86 LC CDR2 option 13 GTKFLVP 87 LC CDR3 option 1 TLWYSNRWV 88 LC CDR3 option 2 ALWYSNRWV 89 LC CDR3 option 3 VLWYDNRWV 90 LC CDR3 option 4 VLWYANRWV 91 LC CDR3 option 5 VLWYSNSWV 92 LC CDR3 option 6 VLWYSNRWI 93 LC CDR3 option 7 VLWYSNRWA 94 Ahth-CD3, clone 2G5 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYALNWVRQAPGKGL EWVARIRSKYNNYATEYADSVKDRFTISRDDSKNTAYLQMNNLKT

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Claims (12)

EDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGG SGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTNFLAPGTPERFSGSLLGGKAALTLSGVQPEDEAE YYCVLWYSNRWAFGGGTKLTVL 95 Ahth-CD3, clone 8A5 EVQLVESGGGLVQPGGSLKLSCAASGFTFNEYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADDVKDRFTISRDDSKNTAYLQMNNLKT EDTAVYYCVRHGNFGNSGISYWAYWGQGTLVTVSSGGGGSGGGG SGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTVGNYPNWVQ QKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWVFGGGTKLTVL 96 Illustrative linker subsequence (GS)n 97 Illustrative linker subsequence (GGS)n 98 Illustrative linker subsequence (GGGS)n 99 Illustrative linker subsequence (GGSG)n 100 Illustrative linker subsequence (GGSGG)n 101 Illustrative linker subsequence (GGGGS)n 102 Illustrative linker subsequence (GGGGG)n 103 Illustrative linker subsequence (GGG)n 104 Illustrative linker subsequence (GGGGS)4 105 6X Histidine HHHHH CLAIM 1. A protein containing a single-chain variable fragment that binds to CD3, containing a heavy chain variable region (VH), a light chain variable region (VL) and a linker, while VH contains complementarity-determining regions VH CDR1, VH CDR2 and VH CDR3, while VL contains complementarity determining regions VL CDR1, VL CDR2 and VL CDR3, where (a) VH CDR1 contains SEQ ID NO: 30, VH CDR2 contains SEQ ID NO: 43, VH CDR3 contains SEQ ID NO: 25, VL CDR1 contains SEQ ID NO: 64, VL CDR2 contains SEQ ID NO: 27 and VL CDR3 contains SEQ ID NO: 89; (b) VH CDR1 contains SEQ ID NO: 23, VH CDR2 contains SEQ ID NO: 24, VH CDR3 contains SEQ ID NO: 55, VL CDR1 contains SEQ ID NO: 67, VL CDR2 contains SEQ ID NO: 77 and VL CDR3 contains SEQ ID NO: 92; (c) VH CDR1 contains SEQ ID NO: 31, VH CDR2 contains SEQ ID NO: 42, VH CDR3 contains SEQ ID NO: 60, VL CDR1 contains SEQ ID NO: 64, VL CDR2 contains SEQ ID NO: 79 and VL CDR3 contains SEQ ID NO: 91; (d) VH CDR1 contains SEQ ID NO: 35, VH CDR2 contains SEQ ID NO: 46, VH CDR3 contains SEQ ID NO: 56, VL CDR1 contains SEQ ID NO: 68, VL CDR2 contains SEQ ID NO: 75, and VL CDR3 contains SEQ ID NO: 28; (e) VH CDR1 contains SEQ ID NO: 32, VH CDR2 contains SEQ ID NO: 47, VH CDR3 contains SEQ ID NO: 56, VL CDR1 contains SEQ ID NO: 65, VL CDR2 contains SEQ ID NO: 80, and VL CDR3 contains SEQ ID NO: 87; (f) VH CDR1 contains SEQ ID NO: 29, VH CDR2 contains SEQ ID NO: 44, VH CDR3 contains SEQ ID NO: 52, VL CDR1 contains SEQ ID NO: 73, VL CDR2 contains SEQ ID NO: 76, and VL CDR3 co- - 34 042731 holds SEQ ID NO: 28; (g) VH CDR1 contains SEQ ID NO: 33, VH CDR2 contains SEQ ID NO: 48, VH CDR3 contains SEQ ID NO: 57, VL CDR1 contains SEQ ID NO: 69, VL CDR2 contains SEQ ID NO: 74 and VL CDR3 contains SEQ ID NO: 28; (h) VH CDR1 contains SEQ ID NO: 38, VH CDR2 contains SEQ ID NO: 24, VH CDR3 contains SEQ ID NO: 25, VL CDR1 contains SEQ ID NO: 62, VL CDR2 contains SEQ ID NO: 81, and VL CDR3 contains SEQ ID NO: 28; (i) VH CDR1 contains SEQ ID NO: 37, VH CDR2 contains SEQ ID NO: 24, VH CDR3 contains SEQ ID NO: 53, VL CDR1 contains SEQ ID NO: 70, VL CDR2 contains SEQ ID NO: 82, and VL CDR3 contains SEQ ID NO: 88; (j) VH CDR1 contains SEQ ID NO: 34, VH CDR2 contains SEQ ID NO: 47, VH CDR3 contains SEQ ID NO: 56, VL CDR1 contains SEQ ID NO: 68, VL CDR2 contains SEQ ID NO: 75, and VL CDR3 contains SEQ ID NO: 28; (k) VH CDR1 contains SEQ ID NO: 29, VH CDR2 contains SEQ ID NO: 24, VH CDR3 contains SEQ ID NO: 54, VL CDR1 contains SEQ ID NO: 71, VL CDR2 contains SEQ ID NO: 83, and VL CDR3 contains SEQ ID NO: 28; (l) VH CDR1 contains SEQ ID NO: 33, VH CDR2 contains SEQ ID NO: 41, VH CDR3 contains SEQ ID NO: 25, VL CDR1 contains SEQ ID NO: 63, VL CDR2 contains SEQ ID NO: 84, and VL CDR3 contains SEQ ID NO: 90; (m) VH CDR1 contains SEQ ID NO: 30, VH CDR2 contains SEQ ID NO: 44, VH CDR3 contains SEQ ID NO: 58, VL CDR1 contains SEQ ID NO: 66, VL CDR2 contains SEQ ID NO: 85, and VL CDR3 contains SEQ ID NO: 28; (n) VH CDR1 contains SEQ ID NO: 40, VH CDR2 contains SEQ ID NO: 45, VH CDR3 contains SEQ ID NO: 56, VL CDR1 contains SEQ ID NO: 26, VL CDR2 contains SEQ ID NO: 78, and VL CDR3 contains SEQ ID NO: 93; or (o) VH CDR1 contains SEQ ID NO: 36, VH CDR2 contains SEQ ID NO: 50, VH CDR3 contains SEQ ID NO: 59, VL CDR1 contains SEQ ID NO: 72, VL CDR2 contains SEQ ID NO: 75 and VL CDR3 contains SEQ ID NO: 28. 2. A protein comprising a CD3 binding single chain variable fragment according to claim 1, wherein said protein has an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 22. 3. Protein containing a single-chain variable fragment that binds to CD3, according to claim 1, and VH CDR1 contains SEQ ID NO: 30, VH CDR2 contains SEQ ID NO: 43, VH CDR3 contains SEQ ID NO: 25, VL CDR1 contains SEQ ID NO: 64, VL CDR2 contains SEQ ID NO: 27 and VL CDR3 contains SEQ ID NO: 89. 4. Protein containing a single-chain variable fragment that binds to CD3, according to claim 1, and VH CDR1 contains SEQ ID NO: 29, VH CDR2 contains SEQ ID NO: 44, VH CDR3 contains SEQ ID NO: 52, VL CDR1 contains SEQ ID NO: 73, VL CDR2 contains SEQ ID NO: 76 and VL CDR3 contains SEQ ID NO: 28. 5. Protein containing a single-chain variable fragment that binds to CD3, according to claim 1, and VH CDR1 contains SEQ ID NO: 29, VH CDR2 contains SEQ ID NO: 24, VH CDR3 contains SEQ ID NO: 54, VL CDR1 contains SEQ ID NO: 71, VL CDR2 contains SEQ ID NO: 83 and VL CDR3 contains SEQ ID NO: 28. 6. Protein containing a single-chain variable fragment that binds to CD3, according to claim 1, and VH CDR1 contains SEQ ID NO: 40, VH CDR2 contains SEQ ID NO: 45, VH CDR3 contains SEQ ID NO: 56, VL CDR1 contains SEQ ID NO: 26, VL CDR2 contains SEQ ID NO: 78 and VL CDR3 contains SEQ ID NO: 93. 7. A protein containing a single-chain variable fragment that binds to CD3, according to claim 1, and the specified protein has an amino acid sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 94 and SEQ ID NO: 95. 8. A protein comprising a CD3-binding single chain variable fragment according to claim 7, said protein having the amino acid sequence shown in SEQ ID NO: 9. 9. A protein containing a single chain variable fragment that binds to CD3 according to claim 7, wherein said protein has the amino acid sequence shown in SEQ ID NO: 14. 10. A protein comprising a CD3 binding single chain variable fragment according to claim 7, said protein having the amino acid sequence shown in SEQ ID NO: 19. 11. A protein containing a single chain variable fragment that binds to CD3 according to claim 7, wherein said protein has the amino acid sequence shown in SEQ ID NO: 94. 12. A protein containing a single-chain variable fragment that binds to CD3, containing a heavy chain variable region (VH), a light chain variable region (VL) and a linker, while -