The hsDC2, an unknown gene, was located on chromosome 4q25. The genetic protein product contained 149 amino acids with the molecular weight of 16.8 kDa. By bioinformatics, three predicted non-membrane portion of hsDC2 gene were designed to immune rabbit. The hsDC2 a1~31and hsDC2 a54~82 gene were inserted in pGEX6p-1 that expressed as fusion protein with GST. The other, hsDC2 a140~149, was synthesized with additional cycteine for conjugation with mcKLH at N-terminal. After four times subcutaneous injection, the antiserum was blooded from rabbits. The antibody was purified from the antiserum by protein A beads and confirmed with antigen by western blotting. The identified antibody was used to examine the expression of hsDC2 protein during U937 differentiation induced by RA and PMA.

HIV infects target cells via fusion events following surface envelope glycoprotein binding to the CD4 receptor and a chemokine co-receptor. Despite the high sequence variability of envelope across and within HIV-1 subtypes, this process requires conserved sequences and structures on gp120, which also represent good targets for HIV-1 neutralizing antibodies. Few examples of HIV-1 broadly neutralizing antibodies exist, but these antibodies may hold the key to a protective HIV-1 vaccine. One such antibody, IgG1 b12 (b12), binds the CD4 binding site on the HIV-1 envelope glycoprotein gp120. To date, no vaccine preparations have been able to elicit a b12-like response. A complete understanding of the mechanism of b12 binding to gp120 is essential to successful design of an b12-like immune response. Until now, strategies to map the b12 binding site on gp120 have utilized indirect techniques and/or core gp120 and have shown that b12 binds to a site on gp120 that overlaps the CD4 binding site. To more directly map the b12 epitope on intact gp120, epitope excision mass spectrometry mapping was carried out in the MALDI QqTOF platform. The putative epitope sequence was confirmed by tandem mass spectrometry sequencing. Epitope mapping revealed a novel binding site for IgG1 b12 at the gp120 amino terminus called Nterm. b12 bound a synthesized peptide of the epitope and the nature of the epitope was explored by ELISA. Although the Nterm epitope is involved in b12-gp120 interactions, ELISAs also show that the epitope does not make up the entire binding site on gp120. Rabbits immunized with a peptide version of the Nterm epitope do express antibodies that bind monomeric gp120, but these antibody responses do not neutralize HIV-1 in vitro. These data indicate that the b12 binding site on gp120 is much more complex than previously thought. The b12 binds the Nterm sequence of gp120, perhaps in conjunction with the CD4 binding site. It has been shown that another HIV-1-neutralizing antibody, 4E10, also binds this novel Nterm epitope, and this may indicate a similar mechanism of action utilized by these two different antibodies. Though not able to elicit neutralizing antibodies on its own, this epitope may be an important element of the neutralizing b12 epitope and an important component of HIV-1 neutralizing antibody responses.

HIV infects target cells via fusion events following surface envelope glycoprotein binding to the CD4 receptor and a chemokine co-receptor. Despite the high sequence variability of envelope across and within HIV-1 subtypes, this process requires conserved sequences and structures on gp120, which also represent good targets for HIV-1 neutralizing antibodies. Few examples of HIV-1 broadly neutralizing antibodies exist, but these antibodies may hold the key to a protective HIV-1 vaccine. One such antibody, IgG1 b12 (b12), binds the CD4 binding site on the HIV-1 envelope glycoprotein gp120. To date, no vaccine preparations have been able to elicit a b12-like response. A complete understanding of the mechanism of b12 binding to gp120 is essential to successful design of an b12-like immune response. Until now, strategies to map the b12 binding site on gp120 have utilized indirect techniques and/or core gp120 and have shown that b12 binds to a site on gp120 that overlaps the CD4 binding site. To more directly map the b12 epitope on intact gp120, epitope excision mass spectrometry mapping was carried out in the MALDI QqTOF platform. The putative epitope sequence was confirmed by tandem mass spectrometry sequencing. Epitope mapping revealed a novel binding site for IgG1 b12 at the gp120 amino terminus called Nterm. b12 bound a synthesized peptide of the epitope and the nature of the epitope was explored by ELISA. Although the Nterm epitope is involved in b12-gp120 interactions, ELISAs also show that the epitope does not make up the entire binding site on gp120. Rabbits immunized with a peptide version of the Nterm epitope do express antibodies that bind monomeric gp120, but these antibody responses do not neutralize HIV-1 in vitro. These data indicate that the b12 binding site on gp120 is much more complex than previously thought. The b12 binds the Nterm sequence of gp120, perhaps in conjunction with the CD4 binding site. It has been shown that another HIV-1-neutralizing antibody, 4E10, also binds this novel Nterm epitope, and this may indicate a similar mechanism of action utilized by these two different antibodies. Though not able to elicit neutralizing antibodies on its own, this epitope may be an important element of the neutralizing b12 epitope and an important component of HIV-1 neutralizing antibody responses.

EG2-hFc is an ≈ 80 kDa chimeric heavy-chain antibody comprised of human IgG1 hinge and fragment crystallisable bivalently linked to EG2; a camelid-derived, heavy chain antibody variable domain specific for the human epithelial growth factor receptor 1 and its associated EGFRvIII mutant. Though previous work revealed EG2-hFc to demonstrate impressive in-vivo tumour accumulation, it’s therapeutic potential, as well as that of chimeric heavy-chain antibodies in general, remains largely unexplored. With this in mind, our current study was successful in showing that EG2-hFc could facilitate in-vitro antibody-dependent cell-mediated cytotoxicity of epithelial growth factor receptor-positive breast cancer cells. Additionally, EG2-hFc’s intrinsic cytotoxicity was augmented following the implementation of engineering strategies that are currently being explored in the context of conventional anti-cancer monoclonal antibodies: including the modification of a conserved N-linked CH2 glycan, as well as the alteration of EG2-hFc’s hinge length. Collectively, these findings contribute to the growing body of research that has revealed chimeric heavy-chain antibodies to be a promising class of novel anti-tumour therapeutics.

This dissertation describes several strategies used to create diversity in non-immune antibody libraries. Two of the strategies were used to create two separate peptide focused libraries. Both of these strategies used to create these antigen-class focused libraries used a single scaffold antibody gene that contained diversity only in the variable heavy region. The scaffold antibody gene one of the libraries, the M:anti-pep library, was chosen based on hypervariable loop canonical structures that are characteristic of other anti-peptide antibodies. Additionally, all of the contact residues of this antibody are commonly used contact residues in other anti-peptide antibodies. These positions and others were varied to incorporate the natural diversity of other anti-peptide antibodies. The second library, the Hu:anti-pep, is based on a widely used, unique combination of human germline antibody segments that express well in bacterial expression. Positions were chosen for variation based on their usage as contact residues in both anti-peptide and anti-protein antibodies. The diversity was less focused than with the M:anti-pep library, incorporating all 20 amino acids at "high usage" positions and only four amino acids at "low usage" positions. Both libraries were validated by phage display selections against the peptide angiotensin (AT) and neuropeptide Y (NPY). The M:anti-pep library yielded specific antibodies to both peptides with dissociation constants as low as 14 nM against AT and 18 nM against NPY. The Hu:anti-pep library yielded specific clones with higher dissociation constants: 49 nM against NPY and 11 [mu]M against AT. The final strategy used to introduce diversity is widely used for affinity maturation of low affinity, previously selected antibodies. Extremely high rates of mutagenesis (2.2% of the gene to 2.7%) were used to create two libraries of the anti-digoxin antibody 26-10. The libraries had been screened by others in an attempt to examine the effects of highrates of mutagenesis on the directed evolution of an antibody. A total of 91 isolated clones from both libraries were sequenced. Several consensus mutations were identified near the CDRH3 in the isolated clones, indicating that they had a positive, selectable effect. This study confirmed that high-error rate antibody libraries contain more active clones than expected. Combinations of the selected consensus mutations from these libraries provide moderate enhancements to the kinetics and expression of the wild-type antibody in a non-synergistic manner. / text

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

Antibody phage display technology mimics the natural immune system, and has been widely used for rapid isolation of single-domain antibodies (sdAbs) with various binding specificities and affinities in the micromolar to low nanomolar range. SdAbs are the variable regions of immunoglobulins (e.g., VH, VL, VHH) and serve as potential probes with therapeutic value. The small size, high solubility, high expression and stability, and high specificity and affinity for the cognate antigen, make sdAbs ideal in improving drug delivery and the overall therapeutic value of antibodies. The main objective of this thesis was to construct a large VL phage display library (~1010 diversity); analyze it via sequence analysis, and to subtractively pan the library for isolation of Neuropilin-1 (NRP1)-specific VLs. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 Semaphorins (Sema3A), contributes to neuron cell death through its interaction with Sema3A in stroke patients. Disruption of this NRP1-Sema3A interaction would allow for axonal outgrowth and neuron regeneration in the area of the brain affected by stroke. Construction of the synthetic phage antibody library utilized a single VL framework with selected positions in the complementarity-determining regions (CDRs) targeted for randomization in vitro using synthetic oligonucleotides that introduced sequence degeneracy. Specific VLs were then selected from the repertoire through subtractive panning against a cell line endogenously expressing NRP1 (PC12) as well as a negative cell line that does not express NRP1 (HEK293) with competitive elution carried out using a synthetic Sema3A-derived peptide. Fifteen VL clones were isolated, cloned in E. coli, expressed and purified, and of these, nine were determined to be non-aggregating by size exclusion chromatography. Further studies will determine the potential therapeutic use of these VL sdAbs as agents in recovery from stroke and neuron degeneration.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.