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The estimated month OS event rates were Median treatment duration was No drug-related deaths occurred in either arm. Adjudicated drug-related interstitial lung disease ILD occurred in Table: LBA1 Summary of results. These data confirm that T-DXd is tolerable with manageable toxicity and a significant improvement in ILD profile vs studies performed in more heavily pretreated pts.

Hoffmann-La Roche Ltd. All other authors have declared no conflicts of interest. This site uses cookies. Some of these cookies are essential, while others help us improve your experience by providing insights into how the site is being used. For more detailed information on the cookies we use, please check our Privacy Policy. Necessary cookies enable core functionality.

The website cannot function properly without these cookies, and you can only disable them by changing your browser preferences. Abstract LBA1. Methods Pts were randomized In these cases, however, the exact locations of conjugation sites remained elusive. Determining the conjugation sites is important, since the affinity of MAb conjugates may be affected significantly by modification of residues in complementarity determining regions CDRs resulting in non-active conjugate species in the mixture.

Recently, we published the structural characterization—primary sequence and posttranslational modifications—of huN, which was performed with tryptic and Asp-N peptide mapping in combination with electrospray ionization time-of-flight mass spectrometry ESI-TOFMS Wang et al. Deglycosylation of huN—DM1 and tryptic and Asp-N protease digestions were performed to help in the characterization. The immunoconjugate, huN—DM1, is a mixture of conjugate species with varying numbers of covalently linked drugs.

The composition of the mixture was analyzed by mass spectrometry of deglycosylated conjugate dghuN—DM1 , which eliminates the complexity in the MS spectrum caused by the heterogeneity in the glycosylation of the CHO cell-derived huN antibody Wang et al. An organic mobile phase was used in the SEC elution to avoid salt interference with protein ionization.

The mass differences between adjacent peaks vary from Da to Da with a mean of Da, which is consistent with the mass of one covalently linked DM1 drug calculated mass increase Da. In a second step, we analyzed the conjugation profiles of the separated dghuN light and heavy chains.

The deconvoluted MS spectra show three and four prominent peaks for the light chain and the heavy chain, respectively. The mass difference between adjacent peaks varies between Da and Da Fig. This moiety is the covalently linked portion of the SPP linker with the free sulfhydryl group, which indicates that DTT reduction of the antibody, as expected, also reduced the disulfide bonds in the drug links.

The number of linkers attached to the light and heavy chains can be obtained directly from the deconvoluted MS spectra Fig. Therefore, both chains of the antibody are modified and conjugated with DM1 drugs. However, the measured masses of the reduced and alkylated light and heavy chains 24, Da and 48, Da differ each from their calculated values 24, Da and 48, Da by more than would be expected as the typical error see above. Based on our previous analysis of the huN antibody, we attribute this difference to incomplete reduction of the intrachain disulfide bonds in both chains Wang et al.

Under such conditions, N -hydroxysuccinimide esters preferentially react with primary amino groups on proteins forming stable amide bonds. Classical peptide mapping techniques were used for the determination of drug conjugation sites using either trypsin or Asp-N protease for digestion of the separated heavy and light chains of reduced and alkylated huN—DM1.

Since DTT reduction removes the DM1 drugs from the conjugate, the modification at the conjugation sites will be the alkylated 4-carboxymethylmercaptooxopentyl linker moiety, which adds Da to the peptide mass. Since modification of a lysine residue will inhibit trypsin cleavage, we compared the tryptic digestion maps of the conjugate heavy and light chains with those from the naked antibody Fig. Some differences in peak intensities and retention times were observed in the light-chain chromatograms from However, MS data showed that none of these peptides were modified, and the cause of these chromatographic differences is not known.

For the heavy chain, a late eluting peak at This peak was assigned by MS analysis to be an uncleaved heavy-chain peptide GF.. K LY whose mass had increased by Da. Thus, it was identified as a modified peptide and the only internal lysine residue, K , was designated a conjugation site. The UV chromatograms at nm of tryptic digests of light and heavy chains from naked antibody and conjugate. The alkylated light and heavy chains were separated by denaturing size exclusion chromatography prior to the enzymatic digestion.

A Naked antibody light chain; B conjugate light chain; C naked antibody heavy chain; D conjugate heavy chain. Because of the high similarity between the chromatograms of conjugated and naked huN heavy- and light-chain tryptic digests, we could not identify further conjugation sites based on differences in their UV traces. Instead, MS data were used to identify modified peptides. First we assumed that linker modification could occur at any lysine residue and calculated the expected mass of all the putative, uncleaved tryptic peptides with a mass increase of Da.

The same peptide masses were also extracted from naked-chain peptide mapping profiles. PK PK would be present with a mass increase of Da calculated peptide mass Peptide HT21 TH.. PK PK contains an internal lysine K , which is not efficiently cleaved by trypsin due to the proline residue C-terminal to it.

This peptide was found to be modified with a linker, since the peptide with an experimental mass of The modification site is likely at residue K , since it is the only modifiable lysine residue in the peptide. A total of 18 modified tryptic peptides were identified; six peptides are from the light chain and 12 are from the heavy chain. The conjugation sites were assigned to the internal lysine residue in each peptide.

All of the sites were found only partially modified, since in each case, the uncleaved, modified peptide and the corresponding two peptides generated through cleavage after the internal lysine residue were identified.

In fact, the unmodified peptides dominated the chromatographic profiles so that the chromatograms of the trypsin digests of the conjugate huN resemble those of the naked antibody Fig. As a result, most modified peptides could not be observed as new peaks in the UV chromatograms due to their low abundances.

Similar to trypsin digestion, only minor differences were observed in the HPLC profiles of Asp-N digests of light and heavy chains from naked antibody and conjugate. However, since fewer peptides are generated in the Asp- N protease digestion, more chromatographic differences caused by conjugation were evident in the chromatograms than in the tryptic digestion maps.

For instance, compared with the naked antibody light-chain profile Fig. The conjugate heavy-chain chromatogram has three new peaks eluting at The observable extra peaks related with antibody conjugation were indicated in the conjugate mapping. The identification of modified heavy-chain peptide HD8 DK.. The unmodified peptide HD8 was found eluting at A new peak was found eluting at The same peptide attached with two linkers at two modification sites was also found, which eluted from 92 to 94 min as a relatively weak EIC peak with a mass increase of Da Fig.

To detect additional modified Asp-N peptides, an analogous approach of data processing was adopted as for the trypsin digest described above. Assuming that modification can occur at each lysine residue, the EICs of putative modified Asp-N peptides were plotted for both naked and conjugated chains, and the data were searched for corresponding peaks.

A peak was assigned to a modified peptide when the mass of the peptide was increased by Da in the conjugate protein maps but not in the corresponding naked protein maps. In Asp-N protease digestion, both modified and unmodified forms of the peptides were found in the conjugate peptide maps, indicating partial modification for all modified peptides. The exact modification sites were generally not assigned for the Asp-N peptides since most of the peptides contain more than one lysine residue.

Additionally, most modified peptides are not fully resolved from other peptides. It is apparent that the two sets of results generally agree with each other; i. KV were not detected in the trypsin digestion.

The total number of identified modification sites increases, therefore, to In addition, the modification at K in the light chain was detected by tryptic mapping but not by the Asp-N mapping. The amino acid sequences of huN light top and heavy bottom chains.

Conjugation sites detected in trypsin digestion are shown in boxes. Asp-N peptides attached with one or two linker moieties are underlined by one or two arrows. The antibody—drug conjugate, HuN—DM1, is prepared by first random modification of the antibody with a cross-linking reagent to which the drug is covalently linked in a second step.

This method yields a conjugate preparation that is heterogeneous at the molecular level. The current study of the structural composition of the conjugate shows that the heterogeneity is twofold. First, the preparation contains several conjugate species that differ in the number of linked drug molecules per antibody molecule: Species with one to six linked drugs were identified, as well as small amounts of unconjugated naked antibody.

Second, each species with a certain number of linked drug molecules can have the linked drugs distributed to different sites of modification: Forty different sites of modification were identified considering that there are two copies of light and heavy chains in each antibody molecule. This number of conjugation sites is large enough so that each site is only partially modified, which leads to low abundances for many modified peptides in the enzymatic digests.

Consequently, such peptides could not be detected in the chromatographic digestion maps. Only careful analysis of the MS data through searching for expected mass peaks allowed the identification of most of the modification sites. It is also worth mentioning that a recent study suggested that tyrosine and histidine residues can also be modified by succinimide esters Leavell et al. However, extensive searches for expected masses indicated that modification of tyrosine and histidine residues were not present in huN—DM1.

This is consistent with the observation that huN—DM1 had retained the full binding affinity of the naked antibody. UV and MS analysis of intact conjugate did show consistent drug load and drug distribution profile from batch to batch data not shown. Under the same reaction conditions, whether or not a lysine residue is modified and to what extent it is modified is expected to depend on the location of the residues in the antibody, which determines the local environment.

For example, one of the modified Asp-N peptides that were detected in the HPLC peptide mapping, HD8 heavy-chain residues — , is located in the hinge region. This may be due to the large solvent exposure and the structural flexibility of the hinge region of the antibody. The solvent accessibility and average depth for the lysine residues in huIgG 1 were calculated, and the B-factors of the side-chain nitrogen of lysine residues were also obtained Fig.

The modified lysine residues in huN—DM1 were mapped to the corresponding lysine residues in huIgG 1. Although this correlation is generally true for most modified lysine residues, exceptions do exist. For example, residues K in the light chain, and K and K in the heavy chain of huIgG 1 are surface residues with relatively large solvent-exposure areas or B-factors Fig.

However, the three lysine residues at similar locations in huN—DM1 were not modified. These exceptions suggest that other factors, such as effects from neighboring residues or hydrogen bonds, may also contribute to the reactivity of individual lysine residues. Residues marked with arrows correspond to the modified lysine residues detected in huN—DM1 labeled with residue numbers in huN The crystal structures of huIgG 1 Fab and Fc were also used to examine the locations of the modified lysine residues in the secondary structures of huN constant regions.

Although they are not located in loop regions, these two residues still have relatively large solvent accessibilities and large B-factors Fig. The two chains are in cyan and yellow and the carbohydrate moieties are in gray. Modified lysine residues in huN—DM1 are mapped to the corresponding residues in huIgG 1 and are shown in red.

In the Fc fragment, the modified lysine residues are only labeled in one chain. The N and C termini are labeled as N-and C-, respectively. The protein images were created using Rasmol V2. In summary, the structural heterogeneity caused by random conjugation of lysine residues in huN— DM1 was characterized by mass spectrometry and peptide mapping.

Twofold heterogeneity was revealed: First, the antibody contains species linked with different number of drug molecules; second, antibody with the same number of drug molecules may have different sites of linkage. The conjugation sites were determined by both trypsin and Asp-N protease digestion.

Although trypsin digestion led to determination of specific sites of modification, chromatographic differences between maps of naked antibody and conjugate appeared more evident upon Asp-N protease digestion. Modification sites detected by both methods are generally consistent, therefore strengthening the results. All modifications are partial, with about seven lysine residues in the light chain and 13 lysine residues in the heavy chain modified. The detected conjugation sites in huN—DM1 were mapped in the crystal structural models of huIgG 1 , and the modified residues were generally found in areas with large solvent accessibility and structural flexibility.

Results of this study have enhanced the understanding of the structure of MAb—maytansinoid conjugates and other types of immunoconjugates. Additionally, correlation of residue location and modification is expected to provide useful information to improve conjugation selectivity and to design conjugates with better structural homogeneity and biological activities. The methods used in this study should be applicable to elucidate structural details of other immunoconjugates.

All other chemicals and enzymes, unless otherwise stated, were purchased from Sigma. Deionized water used was produced by a MilliQ water purification system. The capillary and cone voltages were optimized for maximum sensitivity. MS data were acquired using Masslynx 4. The eluate passed through a UV detector nm and was split before it was directed toward the mass spectrometer. The conjugated antibody eluted at about 7. The column was eluted at a flow rate of 0.

The elution was monitored by UV absorbance at nm and fractions were collected from 9. HCl pH 8. Tryptic digests of naked and conjugated huN light and heavy chains were analyzed with a Jupiter Proteo C 18 column 4.

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Adam kershen A new peak was found eluting at We will get back to you as soon as possible. This site uses cookies. PS Structural characterization of a recombinant monoclonal antibody by electrospray time-of-flight mass spectrometry.
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