Granted Patent B2

US 12,433,954 · App. 17/188,813 · Granted Oct 7, 2025

Inventors: Darrell J. Irvine (Arlington, MA); Karl Dane Wittrup (Boston, MA); Naveen Mehta (Somerville, MA); Leyuan Ma (Brookline, MA); Benjamin Cossette (Durham, NC)

Assignee: Massachusetts Institute of Technology

A61K47/6889A61K40/11A61K40/31A61K40/4211A61K47/543A61P37/04C07K16/2803A61K2239/31A61K2239/38A61K2239/48C07K2317/622C07K2317/92C07K2319/74

Abstract

Claims (114)

1. A method of activating anti-CD19 CAR T cells or increasing proliferation of anti-CD19 CAR T cells in a subject, comprising administering an amphiphilic ligand conjugate, wherein the amphiphilic ligand conjugate comprises:

an anti-CD19 CAR ligand or a multimer thereof; and

a lipid operably linked to the ligand or the multimer,

wherein the anti-CD19 CAR ligand comprises a peptide comprising a sequence motif, wherein the sequence motif binds an anti-CD19 CAR antigen recognition domain, wherein the anti-CD19 CAR antigen recognition domain comprises an antigen recognition domain derived from FMC63, and wherein binding of the sequence motif to the anti-CD19 CAR antigen recognition domain activates a T cell expressing a CAR comprising the anti-CD19 CAR antigen recognition domain, wherein:

(1) the sequence motif comprises the amino acid sequence [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 7), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Ile, Leu, Met, Val, and Arg;

wherein Xaa2 is selected from Ala, Glu, His, Ser, Asp, and Asn;

wherein Xaa3 is selected from Leu, Arg, Ala, Met, Ser, Val, Ile, and Lys;

wherein Xaa4 is selected from Ser, Val, Gln, Ile, Pro, Lys, Glu, and His; and

wherein Xaa5 is selected from Leu, Ile, Arg, His, Gln, and Trp;

(2) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 131), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Leu, Ile, and Val;

wherein Xaa2 is Ser or Lys;

wherein Xaa3 is selected from Arg, Ile, Val, and Met;

wherein Xaa4 is selected from Glu, Lys, and Pro; and

wherein Xaa5 is selected from Leu, Gln, Phe, and Ile;

(3) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 132), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Leu, Ile, and Met;

wherein Xaa2 is selected from Ser, Asn, Asp, and Gly;

wherein Xaa3 is selected from Leu, Met, Ser, Arg, and Lys;

wherein Xaa4 is selected from Glu, Gln, and Pro; and

wherein Xaa5 is Leu or Ile;

(4) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Glu-Leu] (SEQ ID NO: 133), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is Leu or Ile;

wherein Xaa2 is selected from Ser, Asn, and Asp; and

wherein Xaa3 is selected from Gln, Ile, Val, Lys, and Arg; or

(5) the sequence motif comprises an amino acid sequence selected from: RHCPWNCSLL (SEQ ID NO: 5), RICPWSCRAP (SEQ ID NO: 6), and SEQ ID NOs: 10, 12-15, and 20.

2. A method of stimulating an immune response to a target cell population or a target tissue expressing CD19 in a subject, the method comprising administering to the subject anti-CD19 CAR T cells and an amphiphilic ligand conjugate, wherein the amphiphilic ligand conjugate comprises:

an anti-CD19 CAR ligand or a multimer thereof; and

a lipid operably linked to the ligand or the multimer,

wherein the anti-CD19 CAR ligand comprises a peptide comprising a sequence motif, wherein the sequence motif binds an anti-CD19 CAR antigen recognition domain, wherein the anti-CD19 CAR antigen recognition domain comprises an antigen recognition domain derived from FMC63, and wherein binding of the sequence motif to the anti-CD19 CAR antigen recognition domain activates a T cell expressing a CAR comprising the anti-CD19 CAR antigen recognition domain, wherein:

(1) the sequence motif comprises the amino acid sequence [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 7), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Ile, Leu, Met, Val, and Arg;

wherein Xaa2 is selected from Ala, Glu, His, Ser, Asp, and Asn;

wherein Xaa3 is selected from Leu, Arg, Ala, Met, Ser, Val, Ile, and Lys;

wherein Xaa4 is selected from Ser, Val, Gln, Ile, Pro, Lys, Glu, and His; and

wherein Xaa5 is selected from Leu, Ile, Arg, His, Gln, and Trp;

(2) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 131), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Leu, Ile, and Val;

wherein Xaa2 is Ser or Lys;

wherein Xaa3 is selected from Arg, Ile, Val, and Met;

wherein Xaa4 is selected from Glu, Lys, and Pro; and

wherein Xaa5 is selected from Leu, Gln, Phe, and Ile;

(3) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 132), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Leu, Ile, and Met;

wherein Xaa2 is selected from Ser, Asn, Asp, and Gly;

wherein Xaa3 is selected from Leu, Met, Ser, Arg, and Lys;

wherein Xaa4 is selected from Glu, Gln, and Pro; and

wherein Xaa5 is Leu or Ile;

(4) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Glu-Leu] (SEQ ID NO: 133), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is Leu or Ile;

wherein Xaa2 is selected from Ser, Asn, and Asp; and

wherein Xaa3 is selected from Gln, Ile, Val, Lys, and Arg; or

(5) the sequence motif comprises an amino acid sequence selected from: RHCPWNCSLL (SEQ ID NO: 5), RICPWSCRAP (SEQ ID NO: 6), and SEQ ID NOs: 10, 12-15, and 20.

3. A method of inducing an anti-tumor response in a subject with cancer, comprising administering to the subject an amphiphilic ligand conjugate, wherein the subject is receiving or has received anti-CD19 CAR T cells, and wherein the amphiphilic ligand conjugate comprises:

an anti-CD19 CAR ligand or a multimer thereof; and

a lipid operably linked to the ligand or the multimer,

wherein the anti-CD19 CAR ligand comprises a peptide comprising a sequence motif, wherein the sequence motif binds an anti-CD19 CAR antigen recognition domain, wherein the anti-CD19 CAR antigen recognition domain comprises an antigen recognition domain derived from FMC63, and wherein binding of the sequence motif to the anti-CD19 CAR antigen recognition domain activates a T cell expressing a CAR comprising the anti-CD19 CAR antigen recognition domain, wherein:

(1) the sequence motif comprises the amino acid sequence [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 7), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Ile, Leu, Met, Val, and Arg;

wherein Xaa2 is selected from Ala, Glu, His, Ser, Asp, and Asn;

wherein Xaa3 is selected from Leu, Arg, Ala, Met, Ser, Val, Ile, and Lys;

wherein Xaa4 is selected from Ser, Val, Gln, Ile, Pro, Lys, Glu, and His; and

wherein Xaa5 is selected from Leu, Ile, Arg, His, Gln, and Trp;

(2) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 131), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Leu, Ile, and Val;

wherein Xaa2 is Ser or Lys;

wherein Xaa3 is selected from Arg, Ile, Val, and Met;

wherein Xaa4 is selected from Glu, Lys, and Pro; and

wherein Xaa5 is selected from Leu, Gln, Phe, and Ile;

(3) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Xaa4-Xaa5] (SEQ ID NO: 132), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is selected from Leu, Ile, and Met;

wherein Xaa2 is selected from Ser, Asn, Asp, and Gly;

wherein Xaa3 is selected from Leu, Met, Ser, Arg, and Lys;

wherein Xaa4 is selected from Glu, Gln, and Pro; and

wherein Xaa5 is Leu or Ile;

(4) the sequence motif comprises [Arg-Xaa1-Cys-Pro-Trp-Xaa2-Cys-Xaa3-Glu-Leu] (SEQ ID NO: 133), wherein the peptide is capable of forming an intra-peptidyl disulfide bridge;

wherein Xaa1 is Leu or Ile;

wherein Xaa2 is selected from Ser, Asn, and Asp; and

wherein Xaa3 is selected from Gln, Ile, Val, Lys, and Arg; or

(5) the sequence motif comprises an amino acid sequence selected from: RHCPWNCSLL (SEQ ID NO: 5), RICPWSCRAP (SEQ ID NO: 6), and SEQ ID NOs: 10, 12-15, and 20.

4. The method of claim 1 , wherein the anti-CD19 antigen recognition domain comprises an scFv.

5. The method of claim 1 , wherein the two cysteine residues of the sequence motif are capable of forming an intra-peptidyl disulfide bridge.

6. The method of claim 1 , wherein the antigen recognition domain comprises a heavy chain variable sequence (VH) having the sequence of SEQ ID NO: 71 and a light chain variable sequence (VL) having the sequence of SEQ ID NO: 72.

7. The method of claim 1 , wherein the sequence motif comprises an amino acid sequence selected from: RICPWACLSL (SEQ ID NO: 22); RLCPWECRVL (SEQ ID NO: 23); RLCPWACRQL (SEQ ID NO: 24); RLCPWHCAII (SEQ ID NO: 25); RLCPWSCMPR (SEQ ID NO: 26); RLCPWDCLIL (SEQ ID NO: 27); RMCPWSCRPH (SEQ ID NO: 9); RICPWNCSKL (SEQ ID NO: 28); RVCPWSCVEQ (SEQ ID NO: 29); RLCPWNCIHW (SEQ ID NO: 30); or RRCPWSCKKQ (SEQ ID NO: 11).

8. The method of claim 1 , wherein the sequence motif comprises an amino acid sequence selected from: RLCPWKCREL (SEQ ID NO: 31); RLCPWSCIKL (SEQ ID NO: 32); RLCPWSCVEQ (SEQ ID NO: 33); RICPWSCRPL (SEQ ID NO: 34); RLCPWSCIPF (SEQ ID NO: 35); RVCPWSCMPI (SEQ ID NO: 14); and RICPWSCVKQ (SEQ ID NO: 36).

9. The method of claim 1 , wherein the sequence motif comprises RLCPWSCLEI (SEQ ID NO: 37); RICPWSCMEL (SEQ ID NO: 38); RLCPWNCSEL (SEQ ID NO: 39); RLCPWNCRQL (SEQ ID NO: 40); RICPWDCKPI (SEQ ID NO: 41) RMCPWNCREL (SEQ ID NO: 42); RLCPWSCREL (SEQ ID NO: 16); or RICPWGCKEL (SEQ ID NO: 43).

10. The method of claim 1 , wherein the sequence motif comprises RLCPWNCQEL (SEQ ID NO: 44); RICPWSCIEL (SEQ ID NO: 45); RICPWSCVEL (SEQ ID NO: 46); RICPWNCKEL (SEQ ID NO: 18); RLCPWNCREL (SEQ ID NO: 17); RLCPWDCREL (SEQ ID NO: 19); RICPWSCREL (SEQ ID NO: 21); or RLCPWDCKEL (SEQ ID NO: 47).

11. The method of claim 1 , wherein the peptide comprises an amino acid sequence from N-terminus to C-terminus represented by the formula:

[A] x -[M] y -[B] z ;

wherein A, if present, is any amino acid residue, optionally an amino acid residue other than glycine;

wherein M is one or more of the sequence motifs;

wherein B, if present, is any amino acid residue, optionally an amino acid residue other than glycine;

x and z are each integers from 1-20;

y is an integer from 1-5; and

wherein either A is present or B is present, or both A and B are present.

12. The method of claim 11 , wherein (i) A is present and x is 1; (ii) A is present, wherein x is 2-20, and wherein A is the same or different amino acid residue; (iii) wherein B is present and z is 1; (iv) wherein B is present, wherein z is 2-20, and wherein B is the same or different amino acid residue; and/or (v) wherein M is the same or different sequence motifs, and y is 2, 3, 4 or 5.

13. The method of claim 11 , wherein A comprises an amino acid sequence selected from: SAS; GGGSAS (SEQ ID NO: 109); GGSGGGGSAS (SEQ ID NO: 110); GSGGGGSGGGGSAS (SEQ ID NO: 111); GGGGSGGGGSGGGGSAS (SEQ ID NO: 112); PRKHSG (SEQ ID NO: 113); GGGSASPRKHSG (SEQ ID NO: 130); PLS; AKRRERDYVG (SEQ ID NO: 114); PPP; AGT; QFQ; and a combination thereof.

14. The method of claim 11 , wherein (i) B comprises an amino acid sequence represented by the formula:

(SEQ ID NO: 122)

[Tyr-Trp-Leu-Pro-Xaa3-Xaa4],

wherein Xaa3 is any amino acid residue, optionally D or Q; and

wherein Xaa4 is any amino acid residue, optionally E, Q, or R;

(ii) B comprises an amino acid sequence selected from: YWLPQR (SEQ ID NO: 117), YWLPDE (SEQ ID NO: 119), and YWLPDQ (SEQ ID NO: 123); or

(iii) B comprises an amino acid sequence selected from: DNPPFIFGNR (SEQ ID NO: 115), PTPYMMFDM (SEQ ID NO: 116), HPDTRHRIPV (SEQ ID NO: 118), PLDWPW (SEQ ID NO: 120), PSPPRIFGNR (SEQ ID NO: 121); and a combination thereof.

15. The method of claim 11 , wherein the peptide binds to the CAR antigen-recognition domain with (i) an increased binding affinity (K D ) relative to a peptide without either A and/or and B amino acid residues of about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold; and/or (ii) a substantially reduced dissociation rate relative to a peptide without either A and/or B amino acid residues.

16. The method of claim 11 , wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 95-108, and 128-129.

17. The method of claim 1 , wherein the anti-CD19 CAR ligand is operably linked to the lipid via a linker.

18. The method of claim 1 , comprising the multimer of anti-CD19 CAR ligands, wherein the multimer is a dimer, trimer, or tetramer, and wherein the anti-CD19 CAR ligands of the multimer are the same or different.

19. The method of claim 18 , wherein the multimer is a dimer comprising a first anti-CD19 CAR ligand and a second anti-CD19 CAR ligand, wherein the first anti-CD19 CAR ligand and the second anti-CD19 CAR ligand are the same or different, wherein the first anti-CD19 CAR ligand is operably linked to the lipid via a first linker, wherein the second anti-CD19 CAR ligand is operably linked to the lipid via a second linker, and wherein the first linker and the second linker are operably linked to the lipid via a heterotrifunctional compound or lysine.

20. The method of claim 19 , wherein (i) the first linker and the second linker are each selected from: a hydrophilic polymer, a string of hydrophilic amino acids, a polysaccharide, or a combination thereof; and/or (ii) the first linker and the second linker each comprise “N” consecutive polyethylene glycol units, and wherein N is at least 4, 5, 6, 7, 8, 9, or 10.

21. The method of claim 19 , wherein the lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and wherein the first linker and the second linker are a polyethylene glycol polymer selected from PEG4 and PEG8.

22. The method of claim 1 , wherein the amphiphilic ligand is administered in combination with an adjuvant.

23. The method of claim 1 , wherein the lipid is an albumin-binding lipid comprising a hydrophobic tail, wherein the hydrophobic tail is capable of inserting into cell membrane.

Assignments (5)

Continuity (2)

References Cited (24)