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1.
Cell ; 168(6): 1041-1052.e18, 2017 03 09.
Article in English | MEDLINE | ID: mdl-28283060

ABSTRACT

Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems.


Subject(s)
Anaphylaxis/metabolism , Hematopoietic Stem Cells/immunology , Mast Cells/metabolism , Proto-Oncogene Proteins c-kit/metabolism , Signal Transduction , Stem Cell Factor/metabolism , Anaphylaxis/immunology , Animals , Dimerization , Humans , Mast Cells/immunology , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Engineering , Proto-Oncogene Proteins c-kit/agonists , Proto-Oncogene Proteins c-kit/chemistry , Stem Cell Factor/chemistry , Stem Cell Factor/genetics
2.
Proc Natl Acad Sci U S A ; 113(19): E2646-54, 2016 May 10.
Article in English | MEDLINE | ID: mdl-27091975

ABSTRACT

Therapeutic antitumor antibodies treat cancer by mobilizing both innate and adaptive immunity. CD47 is an antiphagocytic ligand exploited by tumor cells to blunt antibody effector functions by transmitting an inhibitory signal through its receptor signal regulatory protein alpha (SIRPα). Interference with the CD47-SIRPα interaction synergizes with tumor-specific monoclonal antibodies to eliminate human tumor xenografts by enhancing macrophage-mediated antibody-dependent cellular phagocytosis (ADCP), but synergy between CD47 blockade and ADCP has yet to be demonstrated in immunocompetent hosts. Here, we show that CD47 blockade alone or in combination with a tumor-specific antibody fails to generate antitumor immunity against syngeneic B16F10 tumors in mice. Durable tumor immunity required programmed death-ligand 1 (PD-L1) blockade in combination with an antitumor antibody, with incorporation of CD47 antagonism substantially improving response rates. Our results highlight an underappreciated contribution of the adaptive immune system to anti-CD47 adjuvant therapy and suggest that targeting both innate and adaptive immune checkpoints can potentiate the vaccinal effect of antitumor antibody therapy.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/administration & dosage , CD47 Antigen/drug effects , CD47 Antigen/immunology , Cancer Vaccines/administration & dosage , Neoplasms, Experimental/immunology , Neoplasms, Experimental/therapy , Adaptive Immunity/drug effects , Adaptive Immunity/immunology , Animals , Antibodies, Monoclonal, Murine-Derived/immunology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/immunology , Cancer Vaccines/immunology , Female , Immunization/methods , Male , Mice , Mice, Inbred C57BL , Molecular Targeted Therapy/methods , Neoplasms, Experimental/pathology , Treatment Outcome
3.
J Biol Chem ; 290(20): 12650-63, 2015 May 15.
Article in English | MEDLINE | ID: mdl-25837251

ABSTRACT

CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.


Subject(s)
Antineoplastic Agents , CD47 Antigen , Macrophages/metabolism , Neoplasms/drug therapy , Phagocytosis/drug effects , Receptors, Immunologic/antagonists & inhibitors , Animals , Antibodies , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , CD47 Antigen/chemistry , CD47 Antigen/genetics , CD47 Antigen/pharmacology , Cell Line, Tumor , Humans , Immunotherapy , Macrophages/pathology , Mice , Neoplasms/genetics , Neoplasms/metabolism , Protein Binding , Protein Engineering , Protein Structure, Tertiary , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism
4.
Science ; 341(6141): 88-91, 2013 Jul 05.
Article in English | MEDLINE | ID: mdl-23722425

ABSTRACT

CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRPα, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRPα variants with about a 50,000-fold increased affinity for human CD47 relative to wild-type SIRPα. As high-affinity SIRPα monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This "one-two punch" directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.


Subject(s)
Adjuvants, Immunologic , Antibodies, Monoclonal/therapeutic use , Antibodies, Neoplasm/therapeutic use , Antigens, Differentiation/therapeutic use , CD47 Antigen/immunology , Neoplasms/therapy , Receptors, Immunologic/therapeutic use , Animals , Antibodies, Monoclonal, Murine-Derived/therapeutic use , Antigens, Differentiation/chemistry , Antigens, Differentiation/genetics , Cell Line, Tumor , Directed Molecular Evolution , Humans , Immunotherapy , Macrophage Activation , Mice , Neoplasms/immunology , Phagocytosis , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Rituximab
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