IgG Subtype Research Articles

Development and Characterization of Peptide Ligands of Immunoglobulin G Fc Region.

Affinity chromatography based on bacterial immunoglobulin (Ig)-binding proteins represents the cornerstone of therapeutic antibody downstream processing. However, there is a pressing need for more robust affinity ligands that would withstand the harsh column sanitization conditions, while still displaying high selectivity for antibodies. Here, we report the development of linear peptide IgG ligands, identified from combinatorial phage-display library screens. The lead peptide was shown to compete with staphylococcal protein A for the IgG Fc region. Trimming analysis and alanine scanning revealed the minimal structural requirements of the peptide for Fc binding, and the minimized peptide GSYWYQVWF recognized all human IgG subtypes. Mutation of glutamine located at the nonessential position 6 to aspartate led to the optimized peptide GSYWYDVWF with 18-fold higher affinity ( KD app. 0.6 μM) compared to the parent peptide. When coupled to paramagnetic beads or a chromatographic matrix, the optimized ligand was shown to selectively enrich antibodies from complex protein mixtures.

Altered Humoral Immune Responses and IgG Subtypes in NOX2-Deficient Mice and Patients: A Key Role for NOX2 in Antigen-Presenting Cells.

Chronic granulomatous disease (CGD) is a primary immunodeficiency resulting from loss of function mutations in the reactive oxygen species generating phagocyte NADPH oxidase (NOX2). CGD patients are prone to infection, but also have an increased susceptibility to autoimmune diseases. The aim of this study was to investigate the role of NOX2 in the regulation of specific immunity. In both CGD patients and NOX2-deficient mice, we observed an alteration in the basal proportions of IgG subtypes. Upon immunization with curdlan—a dectin 1 agonist—NOX2-deficient mice showed increased production of IgG2c compared to controls, and restimulation of lymph node-derived cells led to increased production of IFNγ, but not IL-5, indicative hallmark of an enhanced Th1 response. T cell activation was increased in NOX2-deficient mice and a similar trend was observed in vitro when T cells were co-cultured with NOX2-deficient bone marrow-derived cells. In contrast, no difference in T cell activation was observed when NOX2-deficient T cells were co-cultured with wild-type BMDC. Following stimulation of NOX2-deficient dendritic cells (DCs), no difference in costimulatory molecules was observed, while there was an increase in the release of Th1-driving cytokines. In summary, both CGD patients and CGD mice have an altered IgG subtype distribution, which is associated with an increased IFNγ production. Thus, NOX2 within DCs appears to be an important regulator at the interface of innate and specific immunity, especially after activation of the dectin 1 pathway, limiting immune activation and the development of autoimmunity.

Donor Specific Antibody Shows Different IgG Subtype in Early versus Late Antibody Mediated Renal Allograft Rejection

Background Early and late antibody mediated rejection (EAMR/LAMR) are immunologically distinct entities and represent major risk factors for allograft survival in renal transplantation. The aim of this study was to describe the nature of donor-specific anti-HLA antibodies (DSA) that mediate EAMR and LAMR, and to correlate these data with DSA mean fluorescence intensity (MFI) response to proteasome-inhibitor (PI) therapy. Methods DSA levels (Day 0 and Day 50 therapy) were determined in 35 (11 EAMR + 24 LAMR) patients who received PI-based AMR therapy (1 or two bortezomib cycles +/- a single rituximab dose (375mg/m2)). All patients had at least 9 months of follow-up after AMR therapy. EAMR was defined as occurring within 6 months after transplantation. All donors and recipients had HLA-A,-B,-C,-DRB1, DRB3/4/5,-DQB1, DQA1 and DPB1 typing by molecular methods, and all cases were transplanted after negative prospective flow T- and B-cell crossmatches. AMR was diagnosed using Antibody Working Group and Banff criteria. Anti-HLA donor-specific antibody were identified by Luminex single-antigen beads (One Lambda) using both the generic IgG and IgG1-4 subtype-specific mAb (PE-conjugated, Southern Biotech). Epitope fingerprints were used to assess the therapeutic response (figures). Results 33 DSA were detected in EAMR (11 class I, 22 class II) and 52 DSA in LAMR (11 class I, 41 class II). DSA Class distribution was 43 anti-HLA class I, 27 anti-HLA DR, 50 anti-HLA DQ and 4 anti-HLA DP. Anti-HLA DQ DSAs were found in 3/11 EAMR vs 18/24 LAMR (p=0.01). IgG1 was detected in all patients, IgG2 was detected in 3/11 EAMR and 5/24 LAMR, IgG3 was present in 15/24 LAMR versus 0/11 EAMR (p=0.007), and IgG4 was detected in 2/11 EAMR vs 6/24 LAMR. There were 4/11 EAMR versus 16/24 LAMR patients with multiple IgG subtypes. The DSA MFI reduction at 50 days after PI therapy was 79±22% in EAMR versus 30±26% in LAMR (p=0.00001).. Summary Late AMR demonstrated: 1) a greater number of de novo DSAs; 2) a greater proportion of anti-HLA class II DSA, especially anti-HLA DQ; 3) a higher proportion of IgG3, the subtype with the highest affinity for complement (C1q) binding; 4) a higher degree of multiple IgG subtypes. 5) lesser response in DSA reduction with PI therapy.

Intrastructural Help: Harnessing T Helper Cells Induced by Licensed Vaccines for Improvement of HIV Env Antibody Responses to Virus-Like Particle Vaccines.

ABSTRACTInduction of persistent antibody responses by vaccination is generally thought to depend on efficient help by T follicular helper cells. Since the T helper cell response to HIV Env may not be optimal, we explored the possibility of improving the HIV Env antibody response to virus-like particle (VLP) vaccines by recruiting T helper cells induced by commonly used licensed vaccines to provide help for Env-specific B cells. B cells specific for the surface protein of a VLP can internalize the entire VLP and thus present peptides derived from the surface and core proteins on their major histocompatibility complex class II (MHC-II) molecules. This allows T helper cells specific for the core protein to provide intrastructural help for B cells recognizing the surface protein. Consistently, priming mice with an adjuvanted Gag protein vaccine enhanced the HIV Env antibody response to subsequent booster immunizations with HIV VLPs. To harness T helper cells induced by the licensed Tetanolpur vaccines, HIV VLPs that contained T helper cell epitopes of tetanus toxoid were generated. Tetanol-immunized mice raised stronger antibody responses to immunizations with VLPs containing tetanus toxoid T helper cell epitopes but not to VLPs lacking these epitopes. Depending on the priming immunization, the IgG subtype response to HIV Env after the VLP immunization could also be modified. Thus, harnessing T helper cells induced by other vaccines appears to be a promising approach to improve the HIV Env antibody response to VLP vaccines.IMPORTANCE Induction of HIV Env antibodies at sufficient levels with optimal Fc effector functions for durable protection remains a challenge. Efficient T cell help may be essential to induce such a desirable antibody response. Here, we provide proof of concept that T helper cells induced by a licensed vaccine can be harnessed to provide help for HIV Env-specific B cells and to modulate the Env-specific IgG subtype response.

A Novel Method of Serum Resistance by Escherichia coli That Causes Urosepsis.

ABSTRACTUropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infection, which in some patients can develop into life-threatening urosepsis. Serum resistance is a key virulence trait of strains that cause urosepsis. Recently, we identified a novel method of serum resistance in patients with Pseudomonas aeruginosa lung infections, where patients possessed antibodies that inhibited complement-mediated killing (instead of protecting against infection). These inhibitory antibodies were of the IgG2 subtype, specific to the O-antigen component of lipopolysaccharide (LPS) and coated the bacterial surface, preventing bacterial lysis by complement. As this mechanism could apply to any Gram-negative bacterial infection, we hypothesized that inhibitory antibodies may represent an uncharacterized mechanism of serum resistance in UPEC. To test this, 45 urosepsis patients with paired blood culture UPEC isolates were screened for serum titers of IgG2 specific for their cognate strain’s LPS. Eleven patients had sufficiently high titers of the antibody to inhibit serum-mediated killing of UPEC isolates by pooled healthy control sera. Depletion of IgG or removal of O-antigen restored sensitivity of the isolates to the cognate patient serum. Importantly, the isolates from these 11 patients were more sensitive to killing by serum than isolates from patients with no inhibitory antibodies. This suggests the presence of inhibitory antibodies may have allowed these strains to infect the bloodstream. The high prevalence of patients with inhibitory antibodies (24%) suggests that this phenomenon is an important mechanism of UPEC serum resistance. LPS-specific inhibitory antibodies have now been identified against three Gram-negative pathogens that cause disparate diseases.

Treatment options in myocarditis and inflammatory cardiomyopathy

For myocarditis and inflammatory cardiomyopathy, an etiologically driven treatment is today the best option beyond heart failure therapy. Prerequisites for this are noninvasive and invasive biomarkers including endomyocardial biopsy and polymerase chain reaction on cardiotropic agents. Imaging by Doppler echocardiography and cardiac magnetic resonance imaging as well as cardiac biomarkers such as C‑reactive protein, N‑terminal pro-B-type natriuretic peptide , and troponins can contribute to the clinical work-up of the syndrome but not toward elucidating the underlying cause or pathogenetic process. This review summarizes the phases and clinical features of myocarditis and gives an up-to-date short overview of the current treatment options starting with heart failure and antiarrhythmic therapy. Although inflammation in myocardial disease can resolve spontaneously, often specific treatment directed against the causative agent is required. For fulminant, acute, and chronic autoreactive myocarditis, immunosuppressive treatment has proven to be beneficial in the TIMIC and ESETCID trials; for viral cardiomyopathy and myocarditis, intravenous immunoglobulin IgG subtype and polyvalent intravenous immunoglobulins IgG, IgA, and IgM can frequently resolve inflammation. However, despite the elimination of inflammation, the eradication of parvovirus B19 and human herpesvirus-6 is still a challenge, for which ivIg treatment can become a future key player.

Efficient Humoral and Cellular Immune Responses Induced by a Chimeric Virus-like Particle Displaying the Epitope of EV71 without Adjuvant

ObjectiveTo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant. MethodsThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay. ResultsHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4. ConclusionThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Impact of Payload Hydrophobicity on the Stability of Antibody-Drug Conjugates.

In silico screening of toxin payloads typically employed in ADCs revealed a wide range of hydrophobicities and sizes as measured by log P and topological polar surface area (tPSA) values. These descriptors were used to identify three nontoxic surrogate payloads that encompass the range of hydrophobicity defined by the ADC toxin training set. The uniform drug to antibody ratio (DAR) ADCs were prepared for each surrogate payload by conjugation to the interchain cysteine residues of a model IgG1 subtype mAb. Linkage of these surrogate payloads to a common mAb with a matched DAR value allowed for preliminary analytical interrogation of the influence of payload hydrophobicity on global structure, self-association, and aggregation properties. The results of differential scanning fluorimetry and dynamic light scattering experiments clearly revealed a direct correlation between the destabilization of the native mAb structure and the increasing payload hydrophobicity. Also, self-association/aggregation propensity examined by self-interaction biolayer interferometry or size exclusion HPLC was consistent with increased conversion of the monomeric mAb to higher order aggregated species, with the degree of conversion directly proportional to the payload hydrophobicity. In summary, these findings prove that the payload-dependent structure destabilization and enhanced propensity to self-associate/aggregate driven by the increasing payload hydrophobicity contribute to reduced ADC stability and more complex behavior when assessing exposure and safety/efficacy relationships.

Editors' note: Anti-cytosolic 5′-nucleotidase 1A (cN1A) autoantibodies in motor neuron diseases

Dr. Liewluck recently described 2 patients with motor neuron disease who tested positive for anti-cN1A antibodies by ELISA, an autoantibody reported to be highly specific for inclusion body myositis (IBM). Commenting on this report, Dr. Budhram notes that the anti-cN1A antibody isotype identified in both patients was lacking. He explains that some ELISA tests may identify the presence of immunoglobulin (Ig) G, IgA, or IgM anti-cN1A antibodies, and are potentially somewhat less specific for IBM. He suggests that anti-cN1A antibody isotype data should routinely be included in publications reporting its presence in different patient populations, since this information may provide insight into isotype specificity (or lack thereof) for the diagnosis of IBM. In response, Dr. Liewluck explains that the antibody isotype was not included due to the limited word count and clarifies that the test was done at the commercial laboratory, which detected only the IgG subtype. Dr. Liewluck recently described 2 patients with motor neuron disease who tested positive for anti-cN1A antibodies by ELISA, an autoantibody reported to be highly specific for inclusion body myositis (IBM). Commenting on this report, Dr. Budhram notes that the anti-cN1A antibody isotype identified in both patients was lacking.

Protective efficacy against live Chlamydia muridarum intravaginal challenge in BALB/c mice immunized with MOMP encapsulated PLGA 85/15 nanoparticles

Abstract Chlamydia trachomatis (Ct) remains the leading sexually transmitted bacterial infection worldwide but yet no vaccine to prevent its spread. Attempts to develop vaccines using whole or subunits have failed due to inefficient delivery systems. We have recently developed a Chlamydia nanovaccine by encapsulating its recombinant major outer membrane protein (rMOMP) in poly (lactic acid-co-glycolic acid) (PLGA) 85/15 nanoparticles (PLGA-rMOMP). In the current study, we assessed the systemic and mucosal antibody responses and the protective efficacy of the PLGA-rMOMP nanovaccine against a live C. muridarum (Cm) challenged infection in immunized mice. Female BALB/c mice were immunized subcutaneously three times at two weeks interval with 50 μg each of either PLGA-rMOMP or bare rMOMP and sacrificed two weeks after the last immunization to measure rMOMP-specific systemic and mucosal antibodies. Serum and cervico-vaginal washes were collected to measure IgG including subtypes IgG2a, IgG2b (Th1) and IgG1 (Th2) responses by ELISA. Immunized mice were challenged intravaginally with 105 inclusions forming units (IFUs) of Cm, swabs were collected for three weeks and cultured to quantify bacterial shedding by inoculating McCoy cells. PLGA-rMOMP immunized mice showed enhanced systemic and mucosal IgG antibodies response followed by IgG1 and IgG2b, as compared to bare rMOMP. Moreover, the PLGA-rMOMP immunization induced complete (60% of mice) and partial (40% of mice). Our results show that PLGA-rMOMP stimulates systemic and mucosal antibodies and provides protective efficacy against a Chlamydia infection in mice.

Immunization elicits antigen-specific antibody sequestration in sensory neurons

Abstract The immune and nervous systems coordinate physiological responses to infection and injury. Immunological memory to pathogens is retained in lymphocytes, but the role of neurons in this process is unknown. Here, we report the surprising discovery that peripheral sensory neurons of dorsal root ganglia (DRG) of immunized mice sequester antigen-specific antibodies. A significant increase in the number of total IgG releasing neurons was observed in DRGs isolated from immunized mice as compared to controls (p<0.001). Around 20% of the neurons in DRGs are positive for antibodies. To determine whether sensory neurons synthesize antibodies, DRG neurons from mice immunized with KLH were analyzed by RNA-seq and qPCR. DRG sensory neurons lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity. Transgenic mice that express a reporter fluorescent protein under the control of IgG1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Neural sequestration of antibodies is observed in mice rendered deficient in neuronal Rag2, but not observed in sensory neurons isolated from mice that lack mature B cells (e.g., Rag1 knock out (KO) or μMT mice). Adoptive transfer of Rag1-deficient bone marrow into wild-type mice or wild-type bone marrow into Rag1 KO mice revealed that antibody sequestration is observed in sensory neurons of chimeric mice with wild-type bone marrow but not with Rag1-deficient bone marrow. Our studies demonstrate that DRGs do not synthesize antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. These studies reveal that antigen-specific antibodies and sensory neurons may collaborate in bidirectional communication.

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH)3. Clear differences in the IgG profiles induced by G3, Al(OH)3 or non-adjuvanted vaccine were recorded. Both adjuvants enhanced high and similar levels of the Th2 associated IgG1 subtype compared to mice given vaccine alone. Only G3 enhanced the IgG2a subclass reflecting a Th1 response, whereas Al(OH)3 even abrogated the IgG2a production. Accordingly, G3 enhanced the production of IL-2 and IFN-γ and also of IL-2/IFN-γ double secreting cells, emphasizing the strong Th1 driving effect of G3. Only Al(OH)3 increased splenocyte production of IL-17. Taken together, the results indicate a strong propensity for G3 to induce both Th1 and Th2 driven immune responses.

Memory control by the B cell antigen receptor.

The generation of memory B cells (MBCs) that have undergone immunoglobulin class switching from IgM, which dominates primary antibody responses, to other immunoglobulin isoforms is a hallmark of immune memory. Hence, humoral immunological memory is characterized by the presence of serum immunoglobulins of IgG subtypes known as the γ-globulin fraction of blood plasma proteins. These antibodies reflect the antigen experience of B lymphocytes and their repeated triggering. In fact, efficient protection against a previously encountered pathogen is critically linked to the production of pathogen-specific IgG molecules even in those cases where the primary immune response required cellular immunity, for example, T cell-mediated clearance of intracellular pathogens such as viruses. Besides IgG, also IgA and IgE can provide humoral immunity depending on the microbe's nature and infection route. The molecular mechanisms underlying the preponderance of switched immunoglobulin isotypes during memory antibody responses are a matter of active and controversial debate. Here, we summarize the phenotypic characteristics of distinct MBC subpopulations and discuss the decisive roles of different B cell antigen receptor isotypes for the functional traits of class-switched B cell populations.

Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons.

The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or μMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

Preparation and protection of monoclonal antibody against H7N9 influenza virus hemagglutinin

The first human infection with novel H7N9 influenza virus was reported in March 2013 in China. To date, the H7N9 influenza viruses possess a potential pandemic threat to public health worldwide and have caused severe infection and high mortality in humans in China. The latent period of human infection of H7N9 influenza virus is generally within 7 days. Patients usually show flu like symptoms, such as runny nose, sore throat, fever, cough, headache and muscle pain. Severe patients have rapid development, characterized by severe pneumonia and dyspnea. Antibodies play a major role in protective immunity against the influenza virus infection, and passive immunization with monoclonal antibodies (MAbs) specific to viral proteins might be a potential option for humans against lethal infection of the influenza viruses. It is known that hemagglutinin (HA), is the major viral antigenic protein on the influenza virus particle and the primary target of neutralizing antibodies against influenza virus. In this study, in order to obtain the HA MAbs of influenza virus H7N9 (A/Shanghai/2/2013), study their biology characteristics, and evaluate the prophylactic and therapeutic efficacy of the HA MAbs in mouse model. The BALB/c mice were immunized with inactivated H7N9 vaccine by subcutaneous administration for three times, and boosted with inactivated H7N9 vaccine by the tail vein three days before fusion. Finally, 28 MAbs were obtained and identified by ELISA and hemagglutination inhibition test (HI). Eight IgG subtype MAbs (1A8, 2A12, 2H3, 4A11, 4C8, 4G5, 6A3, 7G2) were selected to identify their biology characteristics. The ELISA titers of ascites of the eight MAbs were 102–106. The eight Mabs exhibited high affinity which can reach nmol/L level and meet the requirement of affinity for monoclonal antibody drug. The immunofluorescence assay indicated that the eight MAbs had excellent specificity for HA protein expressed in 293T cell and were able to recognize the H7N9 influenza virus particles. In addition, all the eight MAbs could inhibit HA activity and the MAbs 2H3, 4A11, 4G5 and 6A3 can also present neutralization activity against the H7N9 live virus. The highest neutralizing titer was up to 1:14125. At the same time, we evaluated the prophylactic and therapeutic efficacy of these MAbs against the homologous H7N9 strain. The activity against H7N9 virus was evaluated by clinical syndromes, survival rates and lung viral titers. The results showed that in the prophylactic and therapeutic experiments, MAb 6A3 can inhibit effectively H7N9 influenza virus infection, provided 100% protection and remarkably reduced the lung viral titers in mouse model. The HA monoclonal antibodies against influenza virus H7N9 were successfully obtained. The HA MAbs not only may be as a prophylactic and therapeutic measure to prevent H7N9 influenza virus infection, but also laid the foundation for us to research the H7N9 HA antigen epitope and develop diagnostic reagents.

Characterization of anti-interferon-γ antibodies in HIV-negative immunodeficient patients infected with unusual intracellular microorganisms.

A major characteristic of immunodeficiency associated with life-threatening intracellular infection in adults is the presence of anti-interferon-γ antibodies. Although little is known about the mechanism underlying this syndrome, it is believed that the antibodies inhibit the activity of downstream signaling pathway of interferon-γ. In this study, the characteristics of these antibodies in patients who presented, or have a history of, intracellular infection and were positive to anti-interferon-γ antibodies were investigated. The antibodies exhibited mainly the IgG1 and the IgG4 subtypes and recognized the C-terminal of the interferon-γ linear epitope containing the KRKR motif, which is required for the biological activity of interferon-γ. The antibodies bound to recombinant interferon-γ with significantly lower avidity than antibodies to a recall antigen, tetanus toxoid, suggesting that the antibodies might have not undergone affinity maturation. The data from this study may provide fundamental information to better understand the properties of anti-interferon-γ antibodies, which can be useful for future studies. Impact statement An increase in the number of immunodeficient patients related to autoantibodies to interferon (IFN)-γ has been observed particularly in East Asian adults. These patients are often presented with opportunistic infections caused by intracellular pathogens, including non-tuberculous mycobacteria (NTM), Cryptococcus neoformans, Penicillium marneffei (now called Talaromyces marneffei), and non-typhoidal Salmonella spp. The mortality rate for this syndrome is relatively high with 32% patients dying at the median time of 25 months after diagnosis. Characterization of these autoantibodies may promote better understanding of the syndrome, an emerging health problem affecting East Asia populations and impeding their welfare and economic development.

Abstract GS3-02: Invasive disease-free survival and gene expression signatures in CALGB (Alliance) 40601, a randomized phase III neoadjuvant trial of dual HER2-targeting with lapatinib added to chemotherapy plus trastuzumab

Abstract Purpose Dual HER2 targeting increases pathologic complete response (pCR) rate to neoadjuvant therapy and improves outcomes in both early and metastatic HER2-positive disease. CALGB 40601 is a randomized phase III trial examining the impact of dual HER2 blockade consisting of trastuzumab (H) and lapatinib (L) added to paclitaxel (T) on pCR, considering tumor and microenvironment molecular features. We previously found that pCR was numerically but not significantly increased with dual therapy, and that tumor molecular subtype and evidence of immune activation significantly and independently affected pCR (Carey et al, JCO 2016). In this secondary analysis, we sought to evaluate the effects of treatment arm and gene expression-defined subgroups on invasive disease free survival (IDFS). Patients and Methods Patients (Pts) with stage II to III HER2-positive breast cancer underwent tumor biopsy followed by random assignment with equal probabilityto paclitaxel plus trastuzumab alone (TH) or with the addition of lapatinib (THL) for 16 weeks before surgery. A paclitaxel plus lapatinib (TL) arm was closed early based on reports of futility from other trials. A secondary endpoint was IDFS, defined as the time from surgery until local or distant recurrence, new primary, or death from any cause, whichever was first. Gene expression signatures were identified by RNA sequencing. Results Between 12/2008 and 2/2012, 305 pts were enrolled. 261 pts had IDFS and gene expression information available (THL, n = 103; TH, n =101; TL, n = 57); there were no significant differences in clinical characteristics between this subset and the entire population. The median IDFS follow-up was 4.6 years with 40 IDFS events having occurred (THL, n=7; TH, n=19; TL, n=14). IDFS was significantly longer in the THL arm compared to standard TH (HR=0.34; 95% CI: 0.14-0.82; p=0.02). IDFS was also significantly longer among pCR than non-pCR pts (HR=0.40; 95% CI: 0.19-0.81; p=0.01), and did not differ by hormone receptor (HR) status, clinical stage, tumor size, race, menopausal status or age. Among gene expression signatures, only immune activation measured by an IgG signature was associated with longer IDFS (HR=0.71; 95% CI: 0.51-0.98; p=0.04); this signature was previously also associated with pCR. Multivariate analysis showed dual therapy (HR=0.35; p=0.02), pCR (HR=0.36; p=0.01), IgG (HR=0.69; p=0.05), and molecular subtype (LumA vs HER2E, HR=0.24, p=0.005) were associated with longer IDFS. A subgroup analysis by hormone receptor status revealed that among pts with HR+ disease, pts with luminal A experienced longer IDFS (HR=0.23; p=0.02) compared to those with luminal B or HER2-enriched molecular subtypes. Conclusion Dual HER2-targeting with lapatinib added to 16 weeks of TH produced significantly longer IDFS than TH alone, despite modest effects on pCR. Similar to pts with HER2-negative disease, pts with luminal A had better IDFS than those with other molecular subtypes. Immune activation as measured by RNA-based signature independently predicted both pCR and IDFS. Support: U10CA180882, U10CA180821, U24CA196171, P50-CA58823, Susan G Komen, BCRF Citation Format: Krop IE, Hillman D, Polley M-Y, Tanioka M, Parker J, Huebner L, Henry NL, Tolaney SM, Dang C, Harris L, Berry DA, Perou CM, Partridge A, Winer EP, Carey LA. Invasive disease-free survival and gene expression signatures in CALGB (Alliance) 40601, a randomized phase III neoadjuvant trial of dual HER2-targeting with lapatinib added to chemotherapy plus trastuzumab [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS3-02.

Safety evaluation of a human chimeric monoclonal antibody that recognizes the extracellular loop domain of claudin-2

Claudin-2 (CLDN-2), a pore-forming tight junction protein with a tetra-transmembrane domain, is involved in carcinogenesis and the metastasis of some cancers. Although CLDN-2 is highly expressed in the tight junctions of the liver and kidney, whether CLDN-2 is a safe target for cancer therapy remains unknown. We recently generated a rat monoclonal antibody (mAb, clone 1A2) that recognizes the extracellular domains of human and mouse CLDN-2. Here, we investigated the safety of CLDN-2-targeted cancer therapy by using 1A2 as a model therapeutic antibody. Because most human therapeutic mAbs are IgG1 subtype that can induce antibody-dependent cellular cytotoxicity, we generated a human–rat chimeric IgG1 form of 1A2 (xi-1A2). xi-1A2 activated Fcγ receptor IIIa in the presence of CLDN-2-expressing cells, indicating that xi-1A2 likely exerts antibody-dependent cellular cytotoxicity. At 24 h after its intravenous injection, xi-1A2 was distributed into the liver, kidney, and tumor tissues of mice bearing CLDN-2-expressing fibrosarcoma cells. Treatment of the xenografted mice with xi-1A2 attenuated tumor growth without apparent adverse effects, such as changes in body weight and biochemical markers of liver and kidney injury. These results support xi-1A2 as the lead candidate mAb for safe CLDN-2-targeted cancer therapy.

Decreased cellular and humoral immunity in patients with breast cancer.

4 Background: A growing body of evidence over the past few years suggests that the presence of immune elements within the tumor or in the tumor stroma has prognostic and predictive value in breast cancer. Immunotherapy is successfully used in many types of cancer, and modulation of immune system became standard part of the cancer patients treatment Responses in breast cancer have been more recently reported. However it has yet to be determined whether predictable biomarkers of response can be identified. Therefore in recent years, research focused on a precise description of the status and function of the immune system.The purpose of the study was to monitor immune responses in patients with breast cancer, particularly the examination of cellular (CD4, CD8, B cells) as well as humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4. It appears that a factor contributing to the immunosupression may be a transforming factor-beta (TGF-beta).It is highly immunosuppressive factor that inhibits the natural and specific immunity against tumors. Methods: 50 patients included in the research project were implemented routine cancer treatment. Basic parameters (histological type and grade, the degree of expression of ER and PR, HER2, and the proliferative marker) were established. Patients were evaluated by a cancer clinical immunologist to exclude immune disorders, allergic or autoimmune origin. Anti-tumor cellular immunity (CD4, CD8, CD19) was measured by flow citometry, humoral immunity (IgG, IgG1, IgG2, IgG3, IgG4) was measured and TGF beta and VEGF production was monitored by ELISA. Results: In breast cancer patients mainly depression in cellular immunity was found. Immunglobuline plasma level was decreased as well (mainly IgG4 subtype). TGF beta as well as VEGF plasma level were increased. Most patients have shown clinical symptoms of immunodeficiency (frequent infections of respiratory or urinary tract, herpetic infections).Those patient could benefit from immunomodulation. Conclusions: The state of anticancer immunity could contribute to the selection of targeted immune therapy in breast cancer patients and to help to find optimal combination of immunotherapy. This project was supported by governmental grant AZV CR 15-28188A.

Immune response to antigen adsorbed to aluminum hydroxide particles: Effects of co-adsorption of ALF or ALFQ adjuvant to the aluminum-antigen complex

Aluminum salts have been used as vaccine adjuvants for >50 years, and they are currently present in at least 146 licensed vaccines worldwide. In this study we examined whether adsorption of Army Liposome Formulation (ALF) to an aluminum salt that already has an antigen adsorbed to it might result in improved immune potency of the aluminum-adsorbed antigen. ALF is composed of a family of anionic liposome-based adjuvants, in which the liposomes contain synthetic phospholipids having dimyristoyl fatty acyl groups, cholesterol and monophosphoryl lipid A (MPLA). For certain candidate vaccines, ALF has been added to aluminum hydroxide (AH) gel as a second adjuvant to form ALFA. Here we show that different methods of preparation of ALF changed the physical structures of both ALF and ALFA. Liposomes containing the saponin QS21 (ALFQ) have also been mixed with AH to form ALFQA as an effective combination. In this study, we first adsorbed one of two different antigens to AH, either tetanus toxoid conjugated to 34 copies of a hapten (MorHap), which has been used in a candidate heroin vaccine, or gp140 protein derived from the envelope protein of HIV-1. We then co-adsorbed ALF or ALFQ to the AH to form ALFA or ALFQA. In each case, the immune potency of the antigen adsorbed to AH was greatly increased by co-adsorbing either ALF or ALFQ to the AH. Based on IgG subtype and cytokine analysis by ELISPOT, ALFA induced predominately a Th2-type response and ALFQ and ALFQA each induced more balanced Th1/Th2 responses.