scholarly journals Preclinical evaluation of immunotherapeutic regimens for fetal/neonatal alloimmune thrombocytopenia

2021 ◽  
Vol 5 (18) ◽  
pp. 3552-3562
Author(s):  
Huiying Zhi ◽  
Maria T. Ahlen ◽  
Björn Skogen ◽  
Debra K. Newman ◽  
Peter J. Newman
Keyword(s):  
Amino Acid ◽  
Amino Acid Polymorphism ◽  
Preclinical Development ◽  
Blocking Antibody ◽  
Central Target ◽  
Female Mice ◽  
Wild Type Female ◽  
Glycoprotein Iiia ◽  
Life Threatening ◽  
Alloimmune Thrombocytopenia

Abstract Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally inherited antigens present on the surface of fetal platelets. The human platelet alloantigen HPA-1a (formerly known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Whites. A single Leu33Pro amino acid polymorphism residing within the ∼50-amino-acid plexin-semaphorin-integrin domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein IIIa [GPIIIa]) is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa and serves as the central target for alloantibody-mediated platelet destruction. To simulate the etiology of human FNAIT, wild-type female mice were pre-immunized with platelets derived from transgenic mice engineered to express the human HPA-1a epitope on a murine GPIIIa backbone. These mice developed a strong alloimmune response specific for HPA-1a, and when bred with HPA-1a+ males, gave birth to severely thrombocytopenic pups that exhibited an accompanying bleeding phenotype. Administering either polyclonal intravenous immunoglobulin G or a human monoclonal blocking antibody specific for the HPA-1a epitope into pregnant female mice resulted in significant elevation of the neonatal platelet count, normalized hemostasis, and prevented bleeding. The establishment of an alloantigen-specific murine model that recapitulates many of the clinically important features of FNAIT should pave the way for the preclinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans.

scholarly journals Prophylactic Administration of Antibodies Specific for the HPA-1a Epitope Prevents Alloimmunization and Platelet Destruction in a Murine Model of Fetal Neonatal Alloimmune Thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3153-3153
Author(s):  
Huiying Zhi ◽  
Douglas Sheridan ◽  
Peter J. Newman
Keyword(s):  
High Titer ◽  
Human Monoclonal Antibody ◽  
Antibody Therapy ◽  
Wild Type ◽  
Amino Acid Polymorphism ◽  
Central Target ◽  
Female Mice ◽  
Wild Type Female ◽  
Life Threatening ◽  
Alloimmune Thrombocytopenia

Abstract Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal alloantibodies directed against paternally inherited antigens present on the surface of fetal and neonatal platelets. The human platelet alloantigen HPA-1a (formerly known as the Pl A1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism near the amino terminus of the integrin b3 subunit (known as GPIIIa in the platelet literature) serves as the central target for alloantibody binding, leading to clearance of both fetal and neonatal platelets, thrombocytopenia, and in the most severe cases, spontaneous- or trauma-induced intracranial hemorrhage. Unlike hemolytic disease of the newborn, which occurs in pregnancies subsequent to parturition-induced alloimmunization, an estimated 25% to 50% of FNAIT cases occur without warning during gestation of the first pregnancy. Though long proposed, there are currently no approved therapies for the prevention of FNAIT. We recently described the development of transgenic mice expressing the human HPA-1a allogeneic epitope on a murine GPIIIa backbone. Transfusion of such platelets into wild-type female mice induced the generation of high-titer anti-HPA-1a alloantibodies that can cross the placenta and recapitulate many of the relevant clinical features of FNAIT. To test the hypothesis that rapid elimination of fetal HPA-1a positive platelets from the circulation of a mother who is HPA-1a negative might prevent maternal alloimmunization and the development of FNAIT, we administered either a hyperimmune plasma-derived polyclonal anti-HPA-1a antibody derived from females having previous cases of FNAIT (termed RLYB211), or a novel human monoclonal antibody directed against the HPA-1a epitope (termed RLYB212), to wild-type female mice prior to challenging them with HPA-1a-positive murine platelets. RLYB211 and RLYB212 were each able to effect the rapid removal of HPA-1a-positive platelets from murine circulation and prevent the development of anti-HPA-1a alloantibodies. Importantly, wild-type female mice pretreated with RLYB211 prior to exposure to HPA-1a-positive platelets, and then impregnated by HPA-1a-positive males, gave birth to HPA-1a-positive pups with significantly improved platelet counts and no bleeding symptoms. These preclinical data establish the potential for prophylactic polyclonal and monoclonal anti-HPA-1a antibody therapy for the prevention of FNAIT in humans. Disclosures Sheridan: Rallybio: Current Employment, Current equity holder in publicly-traded company. Newman: Rallybio: Consultancy, Research Funding.

scholarly journals An Authentic Murine Model of Fetal/Neonatal Alloimmune Thrombocytopenia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 97-97
Author(s):  
Huiying Zhi ◽  
Maria Therese Ahlen ◽  
Trude Rasmussen ◽  
Bjørn Skogen ◽  
Peter J. Newman
Keyword(s):  
Amino Acid ◽  
Transgenic Mice ◽  
Murine Model ◽  
Conflicts Of Interest ◽  
Severe Thrombocytopenia ◽  
Wild Type ◽  
Amino Acid Polymorphism ◽  
Female Mice ◽  
Wild Type Female ◽  
Alloimmune Thrombocytopenia

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies directed against paternally-inherited antigens present on the surface of fetal platelets. The human platelet alloantigen, HPA-1a (also known as the PlA1 alloantigen), is the most frequently implicated HPA for causing FNAIT in Caucasians. A single Leu33Pro amino acid polymorphism residing within the ~50 amino acid plexin/semaphorin/integrin (PSI) domain near the N-terminus of the integrin β3 subunit (platelet membrane glycoprotein (GP)IIIa), is responsible for generating the HPA-1a and HPA-1b epitopes in human GPIIIa. Neither human polyclonal nor mouse monoclonal anti-HPA-1a antibodies, however, recognize murine GPIIIa due to amino acid differences both within and surrounding the HPA-1a alloepitope. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically important platelet alloantigen. We have recently shown that humanizing residues 30, 32, 33, and 39 within the PSI domain, as well as amino acid 470 within the linearly distant but conformationally close Epidermal Growth Factor (EGF) 1 domain, of murine GPIIIa, is sufficient to re-create the epitope recognized by most maternal anti-HPA-1a alloantibodies. We therefore used CRISPR/Cas9 gene editing technology to generate transgenic mice whose platelets express human residues A30P32L33D39Q470 on a murine GPIIIa backbone - hereafter referred to as APLDQ mice. Intraperitoneal injection of an anti-HPA-1a mAb induced severe thrombocytopenia in these APLDQ, but not wild-type, mice. Furthermore, platelets from APLDQ mice, when introduced into wild-type mice, elicited a strong polyclonal immune response that was specific for, and importantly restricted to, the epitopes created by these humanized residues, demonstrating that the APLDQ humanized form of murine GPIIIa is immunogenic in mice. Wild-type female mice pre-immunized with APLDQ platelets, when bred with APLDQ male mice, gave birth to severely thrombocytopenic pups, many of whom exhibited an accompanying bleeding phenotype. Notably, administration of intravenous immunoglobulin G (IVIG) into pregnant female mice at days 10 and 17 lowered the concentration of anti-APLDQ alloantibodies in both the maternal and fetal circulation, and importantly normalized the platelet count in the pups. Taken together, these data establish a novel murine model of FNAIT that recapitulates many of the clinically important features of FNAIT. Availability of APLDQ humanized transgenic mice should pave the way for the pre-clinical development and testing of novel therapeutic and prophylactic modalities to treat or prevent FNAIT in humans. Disclosures No relevant conflicts of interest to declare.

scholarly journals Recreation of Fetal Neonatal Alloimmune Thrombocytopenia in CRISPR/Cas9 Gene-Edited Mice Expressing the Human Platelet Alloantigen, HPA-1a

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 764-764
Author(s):  
Huiying Zhi ◽  
Maria Therese Ahlen ◽  
Bjørn Skogen ◽  
Peter Newman
Keyword(s):  
Amino Acid ◽  
Conflicts Of Interest ◽  
Human Platelet ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Wild Type ◽  
Amino Acid Polymorphism ◽  
Wide Range ◽  
Glycoprotein Iiia ◽  
Alloimmune Thrombocytopenia

Abstract Fetal neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening alloimmune disorder caused by maternal antibodies directed against fetal platelet alloantigens. The human platelet alloantigens, (HPA)-1a/HPA-1b (also known as the PlA1 alloantigen system), residing on the integrin β3 subunit (= platelet membrane glycoprotein IIIa), is the most frequently implicated HPA for causing FNAIT in Caucasians, and a single Leu33Pro amino acid polymorphism is responsible for generating the HPA-1a and HPA-1b epitopes. Neither human nor mouse anti-HPA-1a antibodies recognize murine GPIIIa due to amino acid differences both within and surrounding the polymorphic amino acid, which lies within a long flexible loop in PSI domain of the molecule. As a result, there are currently no authentic mouse models of FNAIT capable of recapitulating the human alloimmune response to this clinically-important platelet antigen. We employed CRISPR/Cas9 gene editing technology to generate mice expressing the human HPA-1a allogeneic epitope. Four critical amino acid substitutions (A30P32L33D39) were introduced into the PSI domain of murine GPIIIa. The resulting humanized form of murine GPIIIa, termed APLD, reacted in a variety of immunoassays with both murine monoclonal (mAb) and human maternal polyclonal antibodies specific for the human HPA-1a epitope. Intraperitoneal injection of an anti-HPA-1a mAb induced thrombocytopenia in APLD, but not wild-type, mice. Importantly, wild-type mice immunized with murine APLD transgenic platelets generated a high-titer HPA-1a-specific antibody response, demonstrating that the humanized form of murine GPIIIa is immunogenic in mice. Finally, pre-immunized female wild-type mice bred with APLD males gave birth to neonatal mice that exhibited profound thrombocytopenia, with the maternal titer directly related to the degree of thrombocytopenia in the neonates. Recapitulating the essential features of FNAIT in mice will facilitate the investigation of a wide-range of clinically-important issues in the diagnosis, treatment, and management of platelet alloimmune disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals A novel murine model of fetal and neonatal alloimmune thrombocytopenia: response to intravenous IgG therapy

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2976-2983 ◽  
Author(s):  
Heyu Ni ◽  
Pingguo Chen ◽  
Christopher M. Spring ◽  
Ebrahim Sayeh ◽  
John W. Semple ◽  
...  
Keyword(s):  
High Titer ◽  
Maternal Antibodies ◽  
Platelet Glycoprotein ◽  
Wild Type ◽  
Wild Type Male ◽  
Glycoprotein Iiia ◽  
Life Threatening ◽  
Β3 Integrin ◽  
Pathogenic Antibodies ◽  
Alloimmune Thrombocytopenia

AbstractFetal and neonatal alloimmune thrombo cytopenia (FNAITP) is a life-threatening bleeding disorder caused by maternal antibodies directed against fetal platelet antigens. The immunoreactive epitopes in FNAITP are primarily located in the extracellular regions of the platelet glycoprotein IIIa (β3 integrin). Here we have established a novel animal model of FNAITP using β3 integrin–deficient (β3-/-) mice. We demonstrated first that these mice are immunoresponsive to β3 integrin; β3-/- mice transfused with wild-type platelets generated specific anti–β3 antibodies which were able to induce thrombocytopenia in wild-type mice. Subsequently, β3-/- female mice (both naive and immunized) were bred with wild-type male mice to recapitulate the features of FNAITP. The titer of generated maternal antibodies correlated with the severity of FNAITP. High titer maternal anti–β3 anti-bodies caused severe fetal thrombocytopenia, intracranial hemorrhage, and even miscarriage. Furthermore, maternal administration of intravenous immunoglobulin G (IgG) ameliorated FNAITP and down-regulated pathogenic antibodies in both the maternal and fetal circulations.

scholarly journals A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1964-1969 ◽  
Author(s):  
F Flug ◽  
R Espinola ◽  
LX Liu ◽  
C SinQuee ◽  
R DaRosso ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Polymorphism ◽  
Glycoprotein Iiia ◽  
Polymerase Chain ◽  
Induced Aggregation

Abstract We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

scholarly journals A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1964-1969
Author(s):  
F Flug ◽  
R Espinola ◽  
LX Liu ◽  
C SinQuee ◽  
R DaRosso ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Polymorphism ◽  
Glycoprotein Iiia ◽  
Polymerase Chain ◽  
Induced Aggregation

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

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