Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.deimmunization | protein engineering I mmunotoxins are chimeric proteins that combine the "magic bullet" specificity of an antibody with the high potency of a toxin. The high specificity of recombinant immunotoxins (RITs) leads to a dramatic decrease in side effects compared with chemotherapy. Moxetumomab Pasudotox (MP) is an RIT that consists of PE38, a fragment of Pseudomonas exotoxin A, fused to an anti-CD22 Fv (1). In a phase I trial for refractory hairy-cell leukemia (HCL), MP had an 86% response rate (2), with 46% complete remissions, and is now in phase III clinical trials (3).Immunogenicity is a stumbling block in the clinical success of many therapeutic proteins (4). Formation of neutralizing antidrug antibodies (5) inactivates the therapeutic agent and can cause serious adverse effects. Although MP had low immunogenicity in the immune-suppressed patients of the HCL trial, some patients did eventually develop antibodies. Consequently, fewer doses could be given to these patients, leading to a reduced response rate. Additionally, RITs targeting solid tumors are less effective than MP because of their high immunogenicity in patients with normal immune systems (6, 7).The role of helper T cells in mounting an immune response is well-established (8, 9). It was previously shown that elimination of murine T-cell epitopes reduced neutralizing antibody formation in mice (10), leading us to the hypothesis that reduction of human T-cell epitopes in the bacterial moiety of RITs would diminish its immunogenicity in humans, allowing more treatment cycles and better antitumor responses, as previously attempted for other therapeutic proteins like erythropoietin (11).To circumvent the immunogenicity of PE38, we previously used peptide pools to map the approximate location of the T-cell epitopes and found an immunodominant and promiscuous epitope that stimulated T cells in 42% of all donors (12). Here, we have done high-resolution mapping of the epitopes ...

Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.mmunogenicity is a major problem in the development of protein therapeutics. Repeated administration of a protein therapeutic can lead to B-cell activation and production of antibodies, rendering the therapeutic clinically ineffective or cross-reacting with host proteins (1). Affinity maturation of antibody-producing memory B cells is initiated by T-cell recognition of peptide epitopes displayed on major histocompatibility complex class II (MHCII) proteins on the surface of mature antigen-presenting cells. Immunogenicity may be reduced by eliminating known T-cell epitopes from the protein sequence and/or increasing the prevalence of sequences already found in the host genome to which T cells would already be tolerant, an approach that has met with substantial clinical success in the humanization of recombinant antibodies (2). However, unlike antibodies, which have been extensively characterized, the mutational tolerance of most proteins is generally not known, and hence, the extension of this approach to proteins of arbitrary structure and function remains a major challenge. Deimmunization efforts have relied, for the most part, on experimental characterization of a large number of point mutants followed by a combination of individual mutations (3, 4).To reduce or eliminate immunogenicity, it would be desirable to have a method that eliminates MHCII-binding epitopes and increases host sequence content without disrupting interactions essential for proper folding and function. The peptide-binding repertoire of many MHCII alleles has been extensively characterized (5), and a number of methods has been developed for predicting the affinity of novel peptides for a given MHCII (6). Coupling of epitope prediction methods with methods for predicting the structural and functional consequences of mutations offers the possibility of reducing the immunogenicity of a target protein without disrupting structure and function. Epitope prediction methods, homolog substitution matrices, and structural stability calculations have been combined to predict optimal epitope-eliminating mutations (7,8). Epitope prediction methods have been integrated with structure-based protein design (9) by combining the 9mer epitope PROPRED matrices with protein design of all residues in a flexible backbone method that allows substantial redesign of protein cores. The combined method was able to eliminate epitope-like sequences while maintaining native-like values for a number of predicted protein stability metrics, but folding, function, ...

Abstract. Lymphocyte proliferation and antibody responses to five peptides corresponding to the N-and C-terminal non-repeat and central repeat regions of Plasmodium falciparum liver-stage antigen-1 (LSA-1) were examined in residents of a highland area of Kenya where malaria transmission is episodic and varies with rainfall. The frequency of lymphocyte proliferation responses (stimulation index > 2) by children (persons Յ 6 years old) and adults (persons Ն 18 years old) was similar and did not differ significantly across seasons. In contrast, the proportion of individuals with IgG antibodies to LSA-1 peptides was higher in the rainy than dry season, and the frequency of these responses was greater for adults than children (39.4% versus 18.7% during the period of high transmission; P ‫ס‬ 0.009). Antibodies to LSA-1 were primarily of the IgG1 and IgG3 subclasses, and these also varied with season (30.1% and 32.5% of individuals had IgG1 and IgG3 in the rainy season versus none and 10.9% in the dry season). There was no significant difference in the time to re-infection between groups of persons with or without IgG antibody or lymphocyte proliferation responses to LSA-1 peptides. These data indicate that age and transmission intensity independently affect IgG antibody responses to LSA-1 but do not influence lymphocyte proliferation in this highland area where malaria transmission is highly variable.

In the design of new enzymes and binding proteins, human intuition is often used to modify computationally designed amino acid sequences prior to experimental characterization. The manual sequence changes involve both reversions of amino acid mutations back to the identity present in the parent scaffold and the introduction of residues making additional interactions with the binding partner or backing up first shell interactions. Automation of this manual sequence refinement process would allow more systematic evaluation and considerably reduce the amount of human designer effort involved. Here we introduce a benchmark for evaluating the ability of automated methods to recapitulate the sequence changes made to computer-generated models by human designers, and use it to assess alternative computational methods. We find the best performance for a greedy one-position-at-a-time optimization protocol that utilizes metrics (such as shape complementarity) and local refinement methods too computationally expensive for global Monte Carlo (MC) sequence optimization. This protocol should be broadly useful for improving the stability and function of designed binding proteins. Proteins 2014; 82:858-866.

Helminth infections in humans and animals are associated with strong T helper 2 (Th2) responses. To determine whether parasite-derived Ag preferentially expand a Th2-like cell population, a filter immunoplaque assay was used to enumerate the frequencies (F0) of PBMC and CD4(+)-enriched PBMC from individuals with helminth infections secreting selected cytokines in response to parasite-derived (PAg) and nonparasite antigens (NPAg). In 20 individuals with lymphatic filariasis, frequency analysis of PBMC secreting IL-4 and IFN-gamma indicated that the F0 of PAg-specific IL-4-secreting cells (geometric mean F0 (GM): 1/12,100) was 57-fold higher than the corresponding F0 of NPAg-reactive cells (GM: 1/692,000; p < 0.02). In marked contrast, the F0 of IFN-gamma-secreting cells responding to PAg (GM: 1/2,700) did not differ from those of cells specific for NAPg (GM: 1/3,400; p = 0.83). In another group of helminth-infected individuals, the F0 of highly enriched CD4+ cells secreting IL-4 and IL-5 in response to PAg (GMs: 1/2,600 and 1/5,600 CD4+ cells, respectively) were also found to be significantly higher than those specific for NPAg (GMs: 1/291,000 and 1/303,000 CD4+; p < 0.05 and p < 0.01, respectively), whereas the corresponding F0 of IFN-gamma- and granulocyte-macrophage-CSF-secreting cells were equivalent for PAg and NPag. Furthermore, the proportion of PAg-specific IL-4- and IL-5-secreting CD4+ cells relative to all cells secreting the given cytokine were approximately 29-fold higher than the proportion of NPAg-specific cells secreting these cytokines. Again, the corresponding proportions of Ag-specific IFN-gamma-and GM-CSF-secreting CD4+ cells were equivalent for PAg and NPAg. Thus, in this ex vivo system, a circulating population of IL-4- and IL-5-secreting (Th2-like) cells has been shown to exist in humans; PAg appears to expand these cells preferentially.