Immunoglobulins and Immunoglobulin Fc Receptors in Nonhuman Primates Commonly Used in Biomedical Research

Georgia State University ScholarWorks @ Georgia State University Biology Dissertations Department of Biology 5-26-2006 Immunoglobulins and Immunoglobulin Fc Receptors in Nonhuman Primates Commonly Used in Biomedical Research Kenneth Alton Rogers Follow this and additional works at: https://scholarworks.gsu.edu/biology_diss Part of the Biology Commons Recommended Citation Rogers, Kenneth Alton, "Immunoglobulins and Immunoglobulin Fc Receptors in Nonhuman Primates Commonly Used in Biomedical Research." Dissertation, Georgia State University, 2006. https://scholarworks.gsu.edu/biology_diss/6 This Dissertation is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. IMMUNOGLOBULINS AND IMMUNOGLOBULIN FC RECEPTORS IN NONHUMAN PRIMATES COMMONLY USED IN BIOMEDICAL RESEARCH by KENNETH ALTON ROGERS Under the Direction of Roberta Attanasio ABSTRACT Antibodies neutralize and eliminate pathogens, malignancies, and toxins by acting either alone or in association with Fc receptors which, once engaged, activate the elimination mechanisms of phagocytic cells. Based on structural differences, antibodies are divided into functionally distinct classes (IgM, IgD, IgG, IgE and IgA). Structure- function relationships within these classes are not well characterized. In addition, animal models for the assessment of potential therapeutic strategies for the modulation of the interaction between antibodies and Fc receptors are not established. Nonhuman primates are widely used to model human diseases and, represent excellent in vivo systems for this assessment. Therefore, we have studied nonhuman primate IgD as well as IgG and IgA specific Fc receptors in rhesus macaques, cynomolgus macaques, baboons and sooty mangabeys. IgD genes had not been identified in nonhuman primates nor the IgD receptors characterized in any species. We characterized IgD genes of the four monkey species, as well as chimpanzees and dogs. In contrast to other antibody classes, the IgD hinge regions are highly conserved between human and nonhuman primates, thus indicating a role in Fc receptor binding. In humans, Fc receptors CD16a (natural killer cells) and CD16b (neutrophils) bind IgG1 and IgG3, and CD89 (myeloid cells) binds IgA. To assess ligand binding and glycosylation properties of nonhuman primate CD16a, CD16b, and CD89, we sequenced, cloned, and generated recombinant molecules in a mammalian expression system. Our results verify the presence of CD16a, but not CD16b in nonhuman primates. CD16a is expressed on monocytes and a subpopulation of lymphocytes. In sooty mangabeys, CD16 is also expressed on neutrophils. Recombinant sooty mangabey/baboon CD16a binds to human IgG1 and IgG2, but not IgG3 and IgG4. Monkey CD89 has the same peripheral blood leukocyte expression profiles as humans, and binds human and recombinant macaque IgA. Blocking of N-glycans inhibited expression of CD89, but only marginally CD16a expression. Although extensive similarities of antibody/Fc receptor interactions exist between human and nonhuman primates, several differences must be considered when evaluating therapeutic strategies. However, these differences can be exploited to further characterize the structure-function relationships existing within antibody molecules and respective receptors. INDEX WORDS: Immunoglobulin, Immunoglobulin Fc receptor, Nonhuman primates, Therapeutic antibodies, Animal models, CD16, CD89 IMMUNOGLOBULINS AND IMMUNOGLOBULIN FC RECEPTORS IN NONHUMAN PRIMATES COMMONLY USED IN BIOMEDICAL RESEARCH by KENNETH ALTON ROGERS A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2006 Copyright by Kenneth Alton Rogers 2006 IMMUNOGLOBULINS AND IMMUNOGLOBULIN FC RECEPTORS IN NONHUMAN PRIMATES COMMONLY USED IN BIOMEDICAL RESEARCH by KENNETH ALTON ROGERS Major Professor: Roberta Attanasio Committee: Phang C. Tai Barbara Baumstark Franco Scinicariello Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2006 iv ACKNOWLEDGMENTS This work is the culmination of the efforts of not only the author, but also a great many other individuals who have contributed. I thank Dr. Roberta Attanasio for providing me with a home for my research, mentorship, and editing of this work. I am grateful for the ceaseless input and endeavors of Dr. Franco Scinicariello to make this project proceed. His optimism in the face of perceived experimental failures was inspiring. Often he would see results where they were scarcely visible beyond the noise and encourage their pursuit. I express my gratitude to my other dissertation committee members Drs. P. C. Tai and Barbara Baumstark. I have great appreciation of my fellow laboratory members, both past and present. Special mention is given to Lakshmi Jayashankar, Feda Masseoud, Jennifer P. Richardson, Doan Nguyen and Nonna Ilybayeva, whom all have contributed their labor for the collection of experimental data presented herein or for other work which did not make the final cut. Finally, I thank my friends and family outside of school for their continuous support of my “career” as a student as well as encouragement to move on to the next career. v TABLE OF CONTENTS ACKOWLEDGEMENTS……………………………………………….. iv LIST OF TABLES………………………………………………………. viii LIST OF FIGURES……………………………………………………... ix LIST OF ABBREVIATIONS…………………………………………… xiii CHAPTER 1 Introduction and Study Objectives………………………… 1 Immunoglobulin (antibody) structure……………………. 7 Immunoglobulin subclasses and functions………………. 13 Immunoglobulin Fc receptors…………………………….. 19 Animal models……………………………………............... 23 Primate evolution…………………………………………. 25 Nonhuman primates in research…………………………. 28 Study objectives…………………………………………… 31 2 Identification and Characterization of Macaque CD89 (IgA Fc Receptor).................................................................... 34 Summary…………………………………………………… 34 Introduction………………………………………………... 35 Materials and methods…………………………………..... 37 Results……………………………………………………… 40 Discussion………………………………………………….. 51 Acknowledgments………………………………………….. 56 vi 3 Immunoglobulin A Fc receptor I (CD89) Homologues in Nonhuman Primates: Identification and Characterization of Baboon and Sooty Mangabey CD89 and Characterization of Rhesus Macaque CD89 Interactions with Human and Rhesus Macaque IgA............................... 57 Summary…………………………………………………. 57 Introduction………………………………………………. 58 Materials and methods………………………………..…. 60 Results…………………………………………………….. 69 Discussion…………………………………………………. 100 Acknowledgements………………………………………. 111 4 Molecular Characterization of IgD in Mammals: Immunoglobulin Heavy Constant Delta (IGHD) Genes in Dogs, Chimpanzees and Four Old World Monkey Species. 112 Summary………………………………………………….. 112 Introduction……………………………………………….. 113 Materials and methods………………………..………….. 118 Results……………………………………………………... 121 Discussion………………………………………………….. 137 Acknowledgements………………………………………... 147 vii 5 Immunoglobulin G (IgG) Fc Receptor III Homologues in Nonhuman Primate Species: Genetic Characterization and Ligand Interactions…………………………………………… 148 Summary……………………………………………………. 148 Introduction…………………………………………………. 149 Materials and methods……………………………..………. 152 Results……………………………………………………….. 160 Discussion…………………………………………………… 179 Acknowledgements…………………………………………. 186 Epilogue: Nonhuman Primate CD16 and Testing of Therapeutic Antibodies……………………………………. 182 6 Conclusions……..……………………………………………. 190 REFERENCES……………………………………………………………. 199 APPENDICES……………………………………………………………. 230 A Supplemental Information Related to the Identification of IgD in Nonhuman Primates and Dogs: Additional Considerations………………………………………………… 230 B IgG Subclass Evolution and Its Implications for IgG Fc Receptor Interactions………………………………………… 249 viii LIST OF TABLES Table 1.1 Functional properties of the human immunoglobulin 15 subclasses……………………………………………………. Table 3.1 Percent identities and divergences of CD89 amino acid 72 sequences……………………………………………………. Table 3.2 Percent identities and divergences for deduced amino acid sequences of CD89 extracellular domain 1, the IgA binding 73 domain…….............................................................................. Table 3.3 Percent identities and divergences for deduced amino acid 74 sequences of CD89 extracellular domain 2………………… Table 3.4 Binding of rhesus macaque CD89 to recombinant rhesus IgA incubated with different concentrations of antigen, NIP (5) 94 BSA…………………………………………………………. Table 4.1 Range of percent identities for deduced amino acid sequences between the IgD heavy chain domains of different mammalian 131 phyla………………………………………………………… Table 6.1 Human and nonhuman primate expression of CD89 and CD16 on different leukocyte populations and antibody subclasses 191 binding patterns of each receptor……………………………. ix LIST OF FIGURES Figure 1.1 Structure of an immunoglobulin molecule………………………. 8 Figure 1.2 Structure of the five mammalian classes of immunoglobulins….. 14 Figure 1.3 Phylogeny of catarrhine primates………………………………... 27 Figure 2.1 Alignment of human and macaque full-length CD89 deduced amino acid sequences……………………………………………. 42 Figure 2.2 Expression of recombinant macaque CD89 on HeLa cells……… 43 Figure 2.3 Alignment of the five rhesus

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