US 2023/0265134 A1 · CH1 DOMAIN VARIANTS ENGINEERED FOR PREFERENTIAL LIGHT CHAIN PAIRING AND MULTISPECIFIC ANTIBODIES COMPRISING THE SAME

A heavy chain constant region 1 (“CH1”) domain variant polypeptide comprising an amino acid substitution at one or more of the following positions:

118, 119, 124, 126-134, 136, 138-143, 145, 147-154, 163, 168, 170-172, 175, 176, 181, 183-185, 187, 190, 191, 197, 201, 203-206, 208, 210-214, 216, and 218, according to EU numbering,
optionally such that the CH1 domain variant polypeptide preferentially pairs;
(i) with a kappa light chain constant region (“CL”) domain as compared to a lambda CL domain and/or with a kappa light chain polypeptide as compared to a lambda light chain polypeptide;
or
(ii) with a lambda CL domain as compared to a kappa CL domain and/or with a lambda light chain polypeptide as compared to a kappa light chain polypeptide;
with the proviso that optionally one or more of the following substitution combinations are excluded;
(a) if residue 141 on CH1 is substituted to C or L, residue 166 is substituted with D or K, residue 128, 129, 162, or 171 on CH1 is substituted to C, and/or residue 147 is substituted to D, said CL does not comprise amino acid substitution;
(b) if position 126 or 220 on CH1 is substituted with valine or alanine, non-cysteine at position 128, 141, or 168 is substituted with cysteine, or CH1 substitutions is L145F, K147A, F170V, S183F, or V185W/F, said CL does not comprise an amino acid substitution
(c) if residue 172 on CH1 is substituted to 172R, residue 174 is mutated to 174G, or residue 190 is substituted to 190M or 190I, that these are not the only CH1 substitution(s);
(d) if the CH1 substitutions consist of L128F, A141I/M/T/L, F170S/A/Y/M, S181M/I/T, S183A/E/K/V and/or V185A/L then CL is not modified;
(e) if the CH1 substitutions consist of 131C/S, 133R/K, 137E/G, 138S/G, 178S/Y, 192N/S, and/or 193F/L, these are not the only CH1 substitutions and/or in a bispecific antibody containing the CH1 domains are of the same human immunoglobulin subtype or allotype;
(f) if the CH1 substitutions consist of 145D/E/R/H/K (IMGT position 26) then there is not a corresponding LC substitution at 129D/E/R/H/K (IMGT position 18);
(g) if the CH1 substitutions consist of 124K/E/R/D there is not a corresponding LC substitution at 176;
(h) if the CH1 substitutions consist of 133V, 150A, 150D, 152D, 173D, and/or 188W, there are not corresponding LC substitutions;
(i) if the CH1 substitutions consist of 133S/W/A, 139W/V/G/I, 143K/E/A, 145E/T/L/Y, 146G, 147T/E, 174V, 175D/R/S, 179K/D/R, 181R, 186R, 188F/L, and/or 190S/A/G/Y there are not corresponding LC substitutions;
(j) if the CH1 substitutions consist of 143A/E/R/K/D and 145T/L there are not corresponding LC substitutions;
(k) if the CH1 substitutions consist of 124A/R/E/W, 145M/T, 143E/R/D/F, 172R/T and 139W/G/C, 179E, and/or 186R, there are not corresponding LC substitutions;
(l) if the CH1 substitutions consist of substituting with cysteine at position 126 127, 128, 134, 141, 171, or 173 then the corresponding LC positions are not modified to form a disulfide bond;
(m) if the CH1 substitutions consist of L145Q, H168A, F170G, S183V, and/or T187E then there are not corresponding kappa or lambda LC substitutions;
(n) if the CH1 substitutions consist of 143D/E, 145T, 190E/D, and/or 124R there are no corresponding CL substitutions;
or
(o) CH1 substitutions consisting of A140C, K147C, and/or S183C there are corresponding CL substitutions;
optionally further comprising one or more amino acid substitutions that increase pairing of a CH1 domain with;
(i) a kappa CL domain as compared to a lambda CL domain;
and/or
(ii) a kappa light chain polypeptide as compared to a lambda light chain polypeptide.

23 more elements

A method of generating a CH1 domain variant library, the method comprising:

(a) providing (i) one or more sets of a polypeptide comprising a CH1 domain paired with a polypeptide comprising a kappa CL domain (“Cκ set”) and/or (ii) one or more sets of a polypeptide comprising a CH1 domain paired with a polypeptide comprising a lambda CL domain (“Cλ set”), optionally wherein the polypeptide comprising a CH1 domain further comprises a heavy chain variable region (VH), further optionally wherein the polypeptide comprising a kappa or a lambda CL domain further comprises a light chain variable region (VL);
(b) selecting one or more amino acid positions of the CH1 domain that are in contact with one or more amino acid positions (i) in the kappa CL domain in the Cκ set, (ii) in the lambda CL domain in the Cλ set, and/or (iii) in the VH in the Cλ set and/or in the Cλ set;
and
(c) producing a library of CH1 domain variant polypeptides or a library of CH1 domain variant-encoding constructs, wherein one or more of the one or more amino acid positions selected in step (b) are substituted with any non-wild-type amino acid,
optionally wherein:
(I) in step (a), said CH1 domain, said kappa CL domain, and said lambda CL domain are wild-type and/or human;
(II) in step (a), both (i) said polypeptide comprising a CH1 domain paired with a polypeptide comprising a kappa CL domain and (ii) said polypeptide comprising a CH1 domain paired with a polypeptide comprising a lambda CL domain are an intact antibody or are an fragment antigen-binding (“Fab);
(III) in step (b), one or more amino acid positions of the CH1 domain is selected if the amino acid residue at said one or more amino acid positions of the CH1 domain have a side-chain atom within a distance of 5 Å of (i) a side-chain atom of the amino acid residue at said one or more amino acid positions in the kappa CL domain, (ii) a side-chain atom of the amino acid residue at said one or more amino acid positions in the lambda CL domain, and/or (iii) a side-chain atom of the amino acid residue at said one or more amino acid positions in the VH;
and/or
(IV) said producing in step (c) is via a degenerate codon, optionally a degenerate RMW codon representing six naturally occurring amino acids (D, T, A, E, K, and N) or a degenerate NNK codon representing all 20 naturally occurring amino acid residues;
wherein further optionally the one or more CH1 amino acid positions selected in step (b) are:
(i) at an interface with the kappa CL domain in at least 10% of a representative set of the Cκ set and has a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of the Cκ set;
(ii) at an interface with the lambda CL domain in at least 10% of a representative set of the Cλ set and has a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of the Cλ set;
and/or
(iii) at an interface with the VH in at least 10% of a representative set of the Cκ and/or Cλ set and has a fractional solvent accessible surface area greater than 10% in at least 90% of a representative set of the Cκ and/or Cλ set.

12 more elements

Ch1 domain variants engineered for preferential light chain pairing and multispecific antibodies comprising the same

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Ch1 domain variants engineered for preferential light chain pairing and multispecific antibodies comprising the same

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Abstract

Claims29