PD1 and/or LAG3 binders
The present invention provides molecules, such as ISVDs and Nanobodies, that bind to PD1 and LAG3 and, optionally to human serum albumin. These molecules have been engineered so as to reduce the incidence of binding by pre-existing antibodies in the bodies of a subject administered such a molecule. Methods for increasing immune response, treating cancer and/or treating an infectious disease with such molecules are provided.
1. A polynucleotide encoding a PD1 binder comprising one or more immunoglobulin single variable domains (ISVDs) that bind PD1, each ISVD comprising (i) a CDR1 comprising the amino acid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); a CDR2 comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4) or VITXSGGITY (SEQ ID NO: 7); and a CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5), and (ii) a substitution of a leucine at position 11 with valine and a substitution of a isoleucine at position 89 with leucine, wherein the positions are numbered according to Kabat, and optionally, further comprising a half-life extender and/or a C-terminal extender.
2. A vector comprising the polynucleotide of claim 1 .
3. A host cell comprising the polynucleotide of claim 1 .
4. A method for making a PD1 binder comprising:
introducing a polynucleotide encoding a PD1 binder comprising one or more immunoglobulin single variable domains (ISVDs) that bind PD1, each ISVD comprising (i) a CDR1 comprising the amino acid sequence IHAMG (SEQ ID NO: 3) or GSIASIHAMG (SEQ ID NO: 6); a CDR2 comprising the amino acid sequence VITXSGGITYYADSVKG (SEQ ID NO: 4) or VITXSGGITY (SEQ ID NO: 7); and a CDR3 comprising the amino acid sequence DKHQSSXYDY (SEQ ID NO: 5), and (ii) a substitution of a leucine at position 11 with valine and a substitution of a isoleucine at position 89 with leucine, wherein the positions are numbered according to Kabat, and optionally, further comprising a half-life extender and/or a C-terminal extender into a host cell and culturing the host cell in a medium under conditions favorable to expression of said PD1 binder from said polynucleotide and, optionally, purifying the PD1 binder from said host cell and/or said medium.
5. The polynucleotide of claim 1 , wherein each ISVD that binds PD1 independently comprises an amino acid sequence selected from the group consisting of
(SEQ ID NO: 57)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSWYDYWGQGTLVTVSS;
(SEQ ID NO: 99)
EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSWYDYWGQGTLVTVSS;
(amino acids 1-119 of SEQ ID NO: 101)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSWYDYWGQGTLVTVSS;
(SEQ ID NO: 103)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITVSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSFYDYWGQGTLVTVSS;
(SEQ ID NO: 104)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSFYDYWGQGTLVTVSS;
and
(SEQ ID NO: 105)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSFYDYWGQGTLVTVSS.
6. The polynucleotide of claim 1 , wherein the polynucleotide further encodes a C-terminal extender, which is an alanine residue.
7. The polynucleotide of claim 1 , wherein the polynucleotide further encodes a half-life extender comprising an ISVD that binds human serum albumin wherein the ISVD comprises a CDR1 comprising the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60) or SFGMS (SEQ ID NO:151); a CDR2 comprising the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 61) or SISGSGSDTL (SEQ ID NO:152); and a CDR3 comprising the amino acid sequence GGSLSR (SEQ ID NO: 62).
8. The polynucleotide of claim 1 , wherein the polynucleotide further encodes a half-life extender comprising an ISVD that binds human serum albumin wherein the ISVD comprises the amino acid sequence
(SEQ ID NO: 142)
EVQLVESGGGVVQPGNSLRLSCAASGETFSSFGMSWVRQAPGKGLEWVSS
ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGG
SLSRSSQGTLVTVSS.
9. The polynucleotide of claim 1 , wherein the polynucleotide encodes (i) an ISVD that binds PD1 comprising the amino acid sequence DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKN TVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS (SEQ ID NO:57); (ii) a half-life extender comprising an ISVD that binds human serum albumin and comprises the amino acid sequence EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKT TLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 142); and, (iii) a C-terminal extender comprising an alanine residue.
10. The polynucleotide of claim 9 , wherein the polynucleotide further encodes a polypeptide linker that links the C-terminus of the ISVD that binds PD1 to the N-terminus of the ISVD that binds human serum albumin, wherein the polypeptide linker comprises the amino acid sequence set forth in SEQ ID NO: 58.
11. The method of claim 4 , wherein each ISVD that binds PD1 independently comprises an amino acid sequence selected from the group consisting of
(SEQ ID NO: 57)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSWYDYWGQGTLVTVSS;
(SEQ ID NO: 99)
EVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSWYDYWGQGTLVTVSS;
(amino acids 1-119 of SEQ ID NO: 101)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITWSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSWYDYWGQGTLVTVSS;
(SEQ ID NO: 103)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITVSGGITYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSFYDYWGQGTLVTVSS;
(SEQ ID NO: 104)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITVSGGITYYADSVKGRFTISRDQSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSFYDYWGQGTLVTVSS;
and
(SEQ ID NO: 105)
DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAV
ITVSGGITYYADSVKGRFTISRDPSKNTVYLQMNSLRPEDTALYYCAGDK
HQSSFYDYWGQGTLVTVSS.
12. The method of claim 4 , wherein the polynucleotide further encodes a C-terminal extender, which is an alanine residue.
13. The method of claim 4 , wherein the polynucleotide further encodes a half-life extender comprising an ISVD that binds human serum albumin wherein the ISVD comprises a CDR1 comprising the amino acid sequence GFTFSSFGMS (SEQ ID NO: 60) or SFGMS (SEQ ID NO:151); a CDR2 comprising the amino acid sequence SISGSGSDTLYADSVKG (SEQ ID NO: 61) or SISGSGSDTL (SEQ ID NO:152); and a CDR3 comprising the amino acid sequence GGSLSR (SEQ ID NO: 62).
14. The method of claim 4 , wherein the polynucleotide further encodes a half-life extender comprising an ISVD that binds human serum albumin wherein the ISVD comprises the amino acid sequence
(SEQ ID NO: 142)
EVQLVESGGGVVQPGNSLRLSCAASGETFSSFGMSWVRQAPGKGLEWVSS
ISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGG
SLSRSSQGTLVTVSS.
15. The method of claim 4 , wherein the polynucleotide encodes (i) an ISVD that binds PD1 comprising the amino acid sequence DVQLVESGGGVVQPGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNSKN TVYLQMNSLRPEDTALYYCAGDKHQSSWYDYWGQGTLVTVSS (SEQ ID NO:57); (ii) a half-life extender comprising an ISVD that binds human serum albumin and comprises the amino acid sequence EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKT TLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 142); and, (iii) a C-terminal extender comprising an alanine residue.
16. The method of claim 15 , wherein the polynucleotide further encodes a polypeptide linker that links the C-terminus of the ISVD that binds PD1 to the N-terminus of the ISVD that binds human serum albumin, wherein the polypeptide linker comprises the amino acid sequence set forth in SEQ ID NO: 58.